Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells

ABSTRACT

Embodiments herein relate to in vitro production methods of hematopoietic stem cell (HSC) and hematopoietic stem and progenitor cell (HSPC) that have long-term multilineage hematopoiesis potentials upon in vivo engraftment. The HSC and HSPCs are derived from pluripotent stem cells-derived hemogenic endothelia cells (HE).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application PCT/US2017/030822 filed on May 3, 2017,which designates the U.S., and which claims the benefit under 35 U.S.C.§ 119(e) of the U.S. Provisional Application No. 62/331,108 filed on May3, 2016, and the U.S. Provisional Application No. 62/468,585 filed onMar. 8, 2017, the contents of each of which are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “701039-087152-PCT_SL.txt”, creation date of Oct. 25, 2018 anda size of 2,646 bytes. The sequence listing submitted via EFS-Web ispart of the specification and is herein incorporated by reference in itsentirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.:R37A1039394, R24DK092760, and UO1-HL100001 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE DISCLOSURE

This disclosure relates to in vitro production methods of hematopoieticstem cell (HSC) and hematopoietic stem and progenitor cell (HSPC)starting from hemogenic endothelia cells (HE) that were induced frompluripotent stem cells, including induced pluripotent stem cells (iPSC),and also relates to long-term multilineage hematopoiesis with theengraftment of these HSCs and HSCPs.

BACKGROUND

There is a lack of supply of functional blood cells for in vivo cellularreplacement therapy, and for in vitro studies of disease modeling, drugscreening, and hematological diseases. Bone marrow transplantation is byfar the most established cellular replacement therapy for a variety ofhematological disorders. The functional unit of a bone marrow transplantis the hematopoietic stem cell (HSC), which resides at the apex of acomplex cellular hierarchy and replenishes blood development throughoutlife. However, the scarcity of HLA-matched HSCs or patient-specific HSCsseverely limits the ability to carry out transplantation, diseasemodeling, drug screening, and in vitro studies of hematologicaldiseases. Often, there is not a large enough cell populationtransplanted to ensure sufficient engraftment and reconstitution invivo.

As such, many studies have been developed to generate HSCs fromalternative sources. For example, reprogramming of somatic cells toinduced pluripotent stem cells (iPSC) has provided access to a widearray of patient-specific pluripotent cells, a promising source fordisease modeling, drug screens and cellular therapies. Pluripotent cellsare induced in human and mouse somatic cells by the forced expression ofthe reprogramming factors: OCT4 (Oct4) and SOX2 (Sox2) with either thecombinations of KLF4 (Klf4) and c-MYC (c-Myc) or NANOG (Nanog) and LIN28(Lin28). Mouse iPS cell lines derived from bone marrow hematopoieticprogenitor cells has been reported. Derivation of human iPS cells frompostnatal human blood cells, from granulocyte colony-stimulating factor(G-CSF) mobilized peripheral blood (PB) CD34+ cells, and from human cordblood (CB) and adult bone marrow (BM) CD34+ cells without anypre-treatment such as G-CSF mobilization has been also reported. TheiPSCs have been shown to differentiate into various cells belonging tothe three germ layers, as demonstrated by the analysis of teratomasgenerated from human and mouse iPS cells. In addition, the pluripotencyof iPS cells is confirmed by the contribution of iPS cell-derived cellsto various organs of the chimeric mice developed from iPScell-introduced blastocysts.

Another approach to generate HSCs from pluripotent stem cells (PSC) isto specify HSCs from its ontogenetic precursors. It is now widelyaccepted that HSCs originate from hemogenic endothelium (HE) in theaorta-gonad-mesonephros (AGM) and arterial endothelium in otheranatomical sites. Recent work on the directed differentiation of HE fromhuman PSC have provided valuable insights into some of the signalingpathways that control the emergence of primitive or definitivepopulations; however, the endothelial-to-hematopoietic transitionremains incompletely understood in human hematopoietic development,making rational intervention challenging. For example, there are reportsof induced definitive HE differentiated from human embryonic bodies (EB)that were derived from iPS cells. However, these HE from PSCs do notengraft in vivo. In contrast, other have shown that real hemogenic cellsfrom human fetal tissues can engraft in mice, indicating missing signalsto confer HSC fate on PSCs.

Therefore, there are still barriers to the generation of HSC from thesealternative sources. In addition to the cell quantity and cell sourceproblems, there is still a hurdle in producing hematopoietic stem andprogenitor cells derived from human pluripotent stem cells (hPSCs) orthe differentiated cells therefrom that would engraft in vivo. Mostly,the primitive HSC and HSPC produced from these alternative sources donot sustain blood production in vivo.

SUMMARY OF THE DISCLOSURE

It is difficult to harvest de novo enough hematopoietic stem cells(HSCs) and hematopoietic stem and progenitor cells (HSPCs) from animalsand humans. It is also difficult to ex vivo culture expand enough ofthese cells for any meaningful therapeutic purposes. Sometimes, the exvivo culture-expanded cells do not differentiate into all thehematopoietic lineage potentials.

Additionally, it is difficult to differentiate HSCs and HSPCs frompluripotent stem cells (PSCs) where the HSCs and HSPCs exhibit all thehematopoietic lineage potentials. One of the most common problems withHSCs and HSPCs derived from PSCs is that the HSCs and HSPCs do notengraft well and in sufficient number in the host after transplantationto sustain blood production in vivo. One of the problems to solve inachieving in vivo long-term multilineage hematopoiesis with theengraftment of these HSCs and HSCPs from the PSCs.

The inventors have found a process to make PSCs-derived HSCs and HSPCsthat would differentiate into all the hematopoietic lineage potentialsand would also engraft well in the host after transplantation so thatthere is sufficient engrafted cells to sustain blood production in vivo.This discovery provides a method for producing functionally relevantHSCs and HSPCs in sufficient quantities for both meaningful experimentaland therapeutic purposes. For example, in vitro experiments, thesePSCs-derived HSCs and HSPCs can be differentiated to the desiredhematopoietic lineage, e.g., erythroid cells, lymphoid cells, andmyeloid cells, for further studies, e.g, drug studies. For example, inin vivo studies, these PSCs-derived HSCs and HSPCs would engraft in ahost, and differentiate into a variety of hematopoietic progeny cells,and reconstitute and populate the circulatory and immune system of thehost.

Embodiments of the present disclosure are based, in part, to thediscovery of a few key transcription factors that would bring about thedifferentiation of HSCs and HSPCs that are derived from pluripotent stemcells derived hemogenic endothelia cells (HE). First, the inventorsshowed that embryonic bodies (EB) are made from pluripotent stem cells,e.g., including induced pluripotent cells. Second, the HE are harvestedfrom the EB, and cultured to induce endothelial-to-hematopoietictransition (EHT) in vitro. Then, the HE cells are transfected withcoding gene sequences of at least the following transcription factors:ERG, HOXA9, HOXA5, LCOR and RUNX1, for the expression of the respectivetranscription factors, thereby to promote differentiation of the HE intoHSCs and HSPCs that exhibit all the hematopoietic lineage potentials.These multilineage HSCs and HSPCs engraft well in recipient host afterimplantation.

Accordingly, in one aspect, provided herein is a method for makinghematopoietic stem cells (HSCs) and hematopoietic stem and progenitorcells (HSPCs) comprising in vitro transfecting hemogenic endotheliacells (HE) with an exogenous gene coding copy of each of the followingtranscription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs that engrafts in recipienthost after implantation. Additional transcription factors, HOXA10 andSPI1, are optionally included.

In another aspect, this disclosure provides is a method of makinghematopoietic stem cells (HSCs) and hematopoietic stem and progenitorcells (HSPCs) comprising (a) generating embryonic bodies (EB) frompluripotent stem cells; (b) isolating hemogenic endothelia cells (HE)from the resultant population of EB; (c) inducingendothelial-to-hematopoietic transition (EHT) in culture in the isolatedHE in order to obtain hematopoietic stem cells, and (d) in vitrotransfecting the induced HE with an exogenous gene coding copy of eachof the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. Additional transcription factors, HOXA10 and SPI1, are optionallyincluded.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprising(a) generating embryonic bodies (EB) from pluripotent stem cells; (b)isolating hemogenic endothelia cells (HE) from the resultant populationof EB; (c) inducing endothelial-to-hematopoietic transition (EHT) inculture in the isolated HE in order to obtain hematopoietic stem cells,and (d) in vitro transfecting the population of HE with an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. Additional transcription factors, HOXA10and SPI1, are optionally included.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is an engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additional transcriptionfactors, HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising (a) generating embryonic bodies (EB)from pluripotent stem cells; (b) isolating hemogenic endothelia cells(HE) from the resultant population of EB; (c) inducingendothelial-to-hematopoietic transition (EHT) in culture in the isolatedHE in order to obtain hematopoietic stem cells, and (d) in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. Additional transcription factors, HOXA10 and SPI1, are optionallyincluded. In some embodiments, this composition is useful for cellularreplacement therapy in a subject. In other embodiments, this compositionis useful for research and laboratory uses. For examples, in drugscreening and testing.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising in vitro transfecting the population ofHE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additionaltranscription factors, HOXA10 and SPI1, are optionally included. In someembodiments, this composition is useful for cellular replacement therapyin a subject. In other embodiments, this composition is useful forresearch and laboratory uses.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells wherein the cells comprise an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. Additional transcription factors, HOXA10and SPI1, are optionally included. Additionally, the engineered cellsfurther comprises reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or NANOG and LIN28.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising (a) generatingembryonic bodies (EB) from pluripotent stem cells; (b) isolatinghemogenic endothelia cells (HE) from the resultant population of EB; (c)inducing endothelial-to-hematopoietic transition (EHT) in culture in theisolated HE in order to obtain hematopoietic stem cells, and (d) invitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included. In some embodiments, this pharmaceuticalcomposition is useful for cellular replacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. Additional transcription factors, HOXA10 and SPI1, are optionallyincluded. In some embodiments, this pharmaceutical composition is usefulfor cellular replacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells and apharmaceutically acceptable carrier, wherein the engineered cellscomprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Additionaltranscription factors, HOXA10 and SPI1, are optionally included.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells are produced by a method comprising(a) generating embryonic bodies (EB) from pluripotent stem cells; (b)isolating hemogenic endothelia cells (HE) from the resultant populationof EB; (c) inducing endothelial-to-hematopoietic transition (EHT) inculture in the isolated HE in order to obtain hematopoietic stem cells,and (d) in vitro transfecting the population of HE with an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. Additional transcription factors, HOXA10and SPI1, are optionally included.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells are produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells to a recipient subject,the population of engineered cells comprise an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. Additional transcription factors, HOXA10 and SPI1, areoptionally included.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE and produced by a method describedherein.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells described herein.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells described hereinand a pharmaceutically acceptable carrier.

In another aspect, this disclosure provides is a pharmaceuticalcomposition described herein for use in cellular replacement therapy ina subject.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a population of engineered cells described, or acomposition described, or a pharmaceutical composition described to arecipient subject.

In another aspect, this disclosure provides is a use of an engineeredcell described herein, a composition comprising of engineered cellsdescribed herein, or a pharmaceutical composition comprising ofengineered cells described herein for the cellular replacement therapyin a subject in need thereof, or for the manufacture of medicament forcellular replacement therapy in a subject in need thereof.

In one embodiment of any one aspect described, the method described isan in vitro method.

In one embodiment of any one aspect described, the EB are generated orinduced from PSC by culturing or exposing the PSC to mophogens for about8 days.

In one embodiment of any one aspect described, the mophogens forgenerating EBs from PSC are selected from the group consisting ofholo-transferrin, mono-thioglycerol (MTG), ascorbic acid, bonemorphogenetic protein (BMP)-4, basic fibroblast growth factor (bFGF),SB431542, CHIR99021, vascular endothelial growth factor (VEGF),interleukin (IL)-6, insulin-like growth factor (IGF)-1, interleukin(IL)-11, stem cell factor (SCF), erythropoietin (EPO), thrombopoietin(TPO), interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand(Flt-3L). The combination of all these factors required to producedefinitive HE.

In one embodiment of any one aspect described, the method describedherein further comprises selecting EBs that are formed from the PSCafter having been exposed or contacted with the described morphogens.

In one embodiment of any one aspect described, the selected EBs are lessthan 800 microns in size and are selected.

In one embodiment of any one aspect described, the EB cells within theselected EBs are compactly adhered to each other and requires trypsindigestion in order to dissociate the cells to individual cells.

In one embodiment of any one aspect described, the EB cells of theselected EBs are dissociated prior to the isolation of HE therefrom.

In one embodiment of any one aspect described, the population of PSCused for generating EBs is induced pluripotent stem cells (iPS cells) orembryonic stem cells (ESC).

In one embodiment of any one aspect described, the iPS cells areproduced by introducing only reprogramming factors OCT4, SOX2, and KLF4,and optionally c-MYC into mature cells. In one embodiment of any oneaspect described, the iPS cells are produced by introducing onlyreprogramming factors OCT4, SOX2, and KLF4, and optionally NANOG andLIN28 into mature cells. The introduction is via any method known in thearts, e.g., viral vectors (AAV, lentiviral, retroviral vectors) that areknown in the art.

In one embodiment of any one aspect described, the mature cells forproducing iPS cells are selected from the group consisting of Blymphocytes (B-cells), T lymphocytes, (T-cells), fibroblasts, andkeratinocytes. Any matured, differentiated cells in the body of amulticellular organism can be used to produce iPCs.

In one embodiment of any one aspect described, the induced pluripotentstem cells are produced by introducing the reprogramming factors once,or two or more times into the mature cells.

In one embodiment of any one aspect described, the disclosedtranscription factors are expressed in the transfected cells, that is,the respective transcription factors: ERG, HOXA9, HOXA5, LCOR, RUNX1,HOXA10, or SPI1, is expressed in the transfected cells.

In another embodiment of any one aspect described, the disclosedtranscription factors are expressed in the engineered cells of thisdisclosure.

In one embodiment of any one aspect described, the expression of thedisclosed transcription factors in the transfected or engineered cellsproduces a population of multi-lineage HSCs and HSPCs.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,engrafts in vivo in the recipient subject and produces blood cells invivo.

In one embodiment of any one aspect described, the population ofmultilineage HSCs and HSPCs, produced by the expression of the disclosedtranscription factors in the transfected or engineered cells,reconstitutes the hematopoietic system in vivo in the recipient subject.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs differentiate to myeloid cells in vivoafter implantation in a host recipient subject. The myeloid cellsproduce MPO upon PMA or cytokine stimulation in vivo.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs differentiate to functional T- and B-cellsin vivo after implantation in a host recipient subject. The functionalT- and B-cells produce IgM and IgG. The functional T- and B-cells alsoundergo immunoglobulin class switching in response to ovalbuminstimulation. The functional T- and B-cells also produces INF-γ.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein express at least one of the following transcriptionfactors: ERG, HOXA9, HOXA5, LCOR, RUNX1, HOXA10, or SPI1, from anexogenous gene encoding the transcription factors in the cells.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+ andCD45+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that are CD34+CD45+ andCD38−.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are CD34+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are are CD34+ and CD45+.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are CD34+CD45+ and CD38−.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that engraft in vivo ina host recipient subject and produce blood cells in vivo.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that reconstitutes thehematopoietic system in vivo when transplanted into a host recipientsubject.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that differentiate tomyeloid cells in vivo, and the myeloid cells produce MPO upon PMA orcytokine stimulation in vivo.

In one embodiment of any one aspect described, the engineered cellsdisclosed herein are multilineage HSCs and HSPCs that differentiate tofunctional T- and B-cells in vivo, the functional T- and B-cells produceIgM and IgG. The functional T- and B-cells also undergo immunoglobulinclass switching in response to ovalbumin stimulation. The functional T-and B-cells also produces INF-γ.

In one embodiment of any one aspect described, the HE are definitive HE.

In one embodiment of any one aspect described, the HE are FLK1+, CD34+,CD43−, and CD235A−. These biomarkers are those on HE before theendothelial-to-hematopoietic transition (EHT).

Definitive HE is a population that is defined by combination of surfaceantigen markers. CD34+FLK1+CD235A−CD43− before EHT.

In one embodiment of any one aspect described, the HE are isolatedimmediately from selected EBs and dissociated EB cells.

In one embodiment of any one aspect described, the HSCs are CD34+.

In one embodiment of any one aspect described, the HSCs are CD34+ andCD45+.

In one embodiment of any one aspect described, the HSPCs are CD34+.

In one embodiment of any one aspect described, the HSPCs are CD34+ andCD45+.

In one embodiment of any one aspect described, the EHT occurs byculturing the isolated HE in thrombopoietin (TPO), interleukin (IL)-3,stem cell factor (SCF), IL-6, IL-11, insulin-like growth factor (IGF)-1,erythropoietin (EPO), vascular endothelial growth factor (VEGF), basicfibroblast growth factor (bFGF), bone morphogenetic protein (BMP)4, Fmsrelated tyrosine kinase 3 ligand (Flt-3L), sonic hedgehog (SHH),angiotensin II, and chemical AGTR1 (angiotensin II receptor type I)blocker losartan potassium.

In one embodiment of any one aspect described, the multilineage HSCsproduced by the methods described in this disclosure areCD34+CD38−CD45+.

In one embodiment of any one aspect described, the multilineage HSPCsproduced by the methods described in this disclosure are CD34+CD45+.

In one embodiment of any one aspect described, the engineered cell ofthis disclosure comprises an exogenous copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1.

In one embodiment of any one aspect described, the engineered cell ofthis disclosure further comprises an exogenous copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.Alternate reprogramming factors in lieu of c-MYC are NANOG and LIN28.

In one embodiment of any one aspect described, the composition ofengineered cells of this disclosure further comprises a pharmaceuticallyacceptable carrier.

In one embodiment of any one aspect described, the subject is a patientwho has undergone chemotherapy or irradiation or both chemotherapy andirradiation, and manifest deficiencies in immune or blood function orlymphocyte reconstitution or both deficiencies in immune function andlymphocyte reconstitution.

In one embodiment of any one aspect described, the subject prior toimplantation, the immune cells are treated ex vivo with prostaglandin E2and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequentengraftment in a recipient subject.

In one embodiment of any one aspect described, the engineered cells ofthis disclosure are autologous to the recipient subject or at least HLAtype matched with the recipient subject.

Definitions

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to methods, and respective componentsthereof as described herein, which are exclusive of any element notrecited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the disclosure.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. A pharmaceutically acceptable carrier will not promote the raisingof an immune response to an agent with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe embodiments of the present disclosure can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Still further, aqueous carriers cancontain more than one buffer salt, as well as salts such as sodium andpotassium chlorides, dextrose, polyethylene glycol and other solutes.Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the methods describedherein that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field of art. Forexample, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

In one embodiment, the “pharmaceutically acceptable” carrier does notinclude in vitro cell culture media.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. Specifically, it refers tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations, andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed. (Mack Publishing Co., 1990). The formulation should suit the mode ofadministration.

A “subject,” as used herein, includes any animal that exhibits a symptomof a monogenic disease, disorder, or condition that can be treated withthe cell-based therapeutics, and methods disclosed elsewhere herein. Inone embodiment, a subject includes any animal that exhibits symptoms ofa disease, disorder, or condition of the hematopoietic system, e.g., ahemoglobinopathy, that can be treated with the cell-based therapeutics,and methods contemplated herein. Suitable subjects (e.g., patients)include laboratory animals (such as mouse, rat, rabbit, or guinea pig),farm animals, and domestic animals or pets (such as a cat or dog).Non-human primates and, preferably, human patients, are included.Typical subjects include animals that exhibit aberrant amounts (lower orhigher amounts than a “normal” or “healthy” subject) of one or morephysiological activities that can be modulated by gene therapy.

In one embodiment, as used herein “treatment” or “treating,” includesany beneficial or desirable effect on the symptoms or pathology of adisease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated. In another embodiment, treatment can involve optionallyeither the reduction or amelioration of symptoms of the disease orcondition, or the delaying of the progression of the disease orcondition. “Treatment” does not necessarily indicate completeeradication or cure of the disease or condition, or associated symptomsthereof.

As used herein, the term “amount” refers to “an amount effective” or “aneffective amount” of transduced therapeutic cells to achieve abeneficial or desired prophylactic or therapeutic result, includingclinical results.

A “prophylactically effective amount” refers to an amount of transducedtherapeutic cells effective to achieve the desired prophylactic result.Typically but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

A “therapeutically effective amount” of transduced therapeutic cells mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the stem and progenitorcells to elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the virus or transduced therapeutic cells are outweighed by thetherapeutically beneficial effects. The term “therapeutically effectiveamount” includes an amount that is effective to “treat” a subject (e.g.,a patient).

As used herein, the terms “administering,” refers to the placement of acomposition or engineered cells of this disclosure into a subject by amethod or route which results in at least a desired effects, forexample, increase number of immune cells or blood cells or platelets.The composition or engineered cells of this disclosure can beadministered by any appropriate route which results in an effectivetreatment in the subject.

As used herein, in one embodiment, the term “hematopoietic stem cell” or“HSC” refers to a stem cell that give rise to all the blood cell typesof the three hematopoietic lineages, erythroid, lymphoid, and myeloid.These cell types include the myeloid lineages (monocytes andmacrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and the lymphoid lineages(T-cells, B-cells, NK-cells). In one embodiment, the term “hematopoieticstem cell” or “HSC” refers to a stem cell that have the following cellsurface markers: CD34+, CD59+, Thy1/CD90+, CD38lo/−, and C-kit/CD117+.In one embodiment, the term “hematopoietic stem cell” or “HSC” refers toa stem cell that is at least CD34+. In one embodiment, the term“hematopoietic stem cell” or “HSC” refers to a stem cell that is atleast CD34+ and C-kit/CD117+. In another embodiment, the term HSC refersto a stem cell that is at least CD34+. In another embodiment, the termHSC refers to a stem cell that is at least CD34+/CD45+. In anotherembodiment, the term HSC refers to a stem cell that is at leastCD34+/CD45+/CD38−.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and refers to a pluripotent cell artificiallyderived by the transfection of following reprogramming factors OCT4,SOX2, KLF4 and optionally c-MYC or optionally NANOG and LIN28, into afrom a differentiated cell.

As used herein, the term “lineage” when used in the context of stem andprogenitor cell differentiation and development refers to the celldifferentiation and development pathway which the cell can take tobecoming a fully differentiated cell. For example, a HSC has threehematopoietic lineages, erythroid, lymphoid, and myeloid; the HSC hasthe potential, ie., the ability, to differentiate and develop into thoseterminally differentiated cell types known for all these three lineages.When the term “multilineage” used, it means the cell is able in thefuture differentiate and develop into those terminally differentiatedcell types known for more than one lineage. For example, the HSC hasmultilineage potential. When the term “limited lineage” used, it meansthe cell can differentiate and develop into those terminallydifferentiated cell types known for one lineage. For example, a commonmyeloid progenitor cell or a megakaryocyte-erythroid progenitor has alimited lineage because the cell can only differentiate and develop intothose terminally differentiated cell types of the myeloid lineage andnot that of the lymphoid lineage. Terminally differentiated cells of themyeloid lineage include erythrocytes, monocytes, macrophages,megakaryocytes, myeloblasts, dendritic cells, and granulocytes(basophils, neutrophils, eosinophils, and mast cells); and terminallydifferentiated cells of the lymphoid lineage include T lymphocytes/Tcells, B lymphocytes/B cells, and natural killer cells.

As used herein, the term “a progenitor cell” refers to an immature orundifferentiated cell that has the potential later on to mature(differentiate) into a specific cell type, for example, a blood cell, askin cell, a bone cell, or a hair cells. Progenitor cells have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate. A progenitor cell also canproliferate to make more progenitor cells that are similarly immature orundifferentiated.

As used herein, the term “multilineage hematopoietic progenitor cells”or “hematopoietic stem and progenitor cells (HSPC)” refer tohematopoietic cells (cell that form the blood) that have the ability orpotential to generate, or differentiate into, multiple types ofhematopoietic lineage cells.

As used herein, the term “long-term” when used in the context of“multilineage hematopoiesis” refers to in vivo implanted HSCs and/orHSPCs being capable of producing the three hematopoietic lineage cells,erythroid, lymphoid, and myeloid, for up to at least 12 weeks posttransplantation.

As used herein, the term “multilineage hematopoiesis” in the context ofHSCs and/or HSPCs refers to these cells capable of producing at leastthe three hematopoietic lineage cells, erythroid, lymphoid, and myeloid.

The term “differentiated cell” is meant any primary cell that is not, inits native form, pluripotent as that term is defined herein. The term a“differentiated cell” also encompasses cells that are partiallydifferentiated, such as multipotent cells (e.g. adult somatic stemcells). In some embodiments, the term “differentiated cell” also refersto a cell of a more specialized cell type derived from a cell of a lessspecialized cell type (e.g., from an undifferentiated cell or areprogrammed cell) where the cell has undergone a cellulardifferentiation process.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term meaning a “differentiated cell” isa cell that has progressed further down the developmental pathway thanits precursor cell. Thus in some embodiments, a reprogrammed cell asthis term is defined herein, can differentiate to lineage-restrictedprecursor cells (such as a mesodermal stem cell or a endodermal stemcell), which in turn can differentiate into other types of precursorcells further down the pathway (such as an tissue specific precursor,for example, a cardiomyocyte precursor, or a pancreatic precursoe), andthen to an end-stage differentiated cell, which plays a characteristicrole in a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of muiltipotent cells include adult somatic stemcells, such as for example, hematopoietic stem cells and neural stemcells, hair follicle stem cells, liver stem cells etc. Multipotent meansa stem cell may form many types of cells in a given lineage, but notcells of other lineages. For example, a multipotent blood stem cell canform the many different types of blood cells (red, white, platelets,etc. . . . ), but it cannot form neurons; cardiovascular progenitor cell(MICP) differentiation into specific mature cardiac, pacemaker, smoothmuscle, and endothelial cell types; pancreas-derived multipotentprogenitor (PMP) colonies produce cell types of pancreatic lineage(cells that produces insulin, glucagon, amylase or somatostatin) andneural lineage (cells that are morphologically neuron-like,astrocytes-like or oligodendrocyte-like).

The term a “reprogramming gene”, as used herein, refers to a gene whoseexpression, contributes to the reprogramming of a differentiated cell,e.g. a somatic cell to an undifferentiated cell (e.g. a cell of apluripotent state or partially pluripotent state). A reprogramming genecan be, for example, genes encoding master transcription factors Sox2,Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like. The term “reprogrammingfactor” refers to the protein encoded by the reprogramming gene.

The term “exogenous” refers to a substance present in a cell other thanits native source. The terms “exogenous” when used herein refers to anucleic acid (e.g. a nucleic acid encoding a reprogramming transcriptionfactor, e.g. Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like) or aprotein (e.g., a transcription factor polypeptide) that has beenintroduced by a process involving the hand of man into a biologicalsystem such as a cell or organism in which it is not normally found orin which it is found in lower amounts. A substance (e.g. a nucleic acidencoding a sox2 transcription factor, or a protein, e.g., a SOX2polypeptide) will be considered exogenous if it is introduced into acell or an ancestor of the cell that inherits the substance.

The term “isolated” as used herein signifies that the cells are placedinto conditions other than their natural environment. The term“isolated” does not preclude the later use of these cells thereafter incombinations or mixtures with other cells.

As used herein, the term “expanding” refers to increasing the number oflike cells through cell division (mitosis). The term “proliferating” and“expanding” are used interchangeably.

As used herein, a “cell-surface marker” refers to any molecule that isexpressed on the surface of a cell. Cell-surface expression usuallyrequires that a molecule possesses a transmembrane domain. Somemolecules that are normally not found on the cell-surface can beengineered by recombinant techniques to be expressed on the surface of acell. Many naturally occurring cell-surface markers are termed “CD” or“cluster of differentiation” molecules. Cell-surface markers oftenprovide antigenic determinants to which antibodies can bind to. Acell-surface marker of particular relevance to the methods describedherein is CD34. The useful hematopoietic progenitor cells according tothe present disclosure preferably express CD34 or in other words, theyare CD34 positive.

A cell can be designated “positive” or “negative” for any cell-surfacemarker, and both such designations are useful for the practice of themethods described herein. A cell is considered “positive” for acell-surface marker if it expresses the marker on its cell-surface inamounts sufficient to be detected using methods known to those of skillin the art, such as contacting a cell with an antibody that bindsspecifically to that marker, and subsequently performing flow cytometricanalysis of such a contacted cell to determine whether the antibody isbound the cell. It is to be understood that while a cell may expressmessenger RNA for a cell-surface marker, in order to be consideredpositive for the methods described herein, the cell must express it onits surface. Similarly, a cell is considered “negative” for acell-surface marker if it does not express the marker on itscell-surface in amounts sufficient to be detected using methods known tothose of skill in the art, such as contacting a cell with an antibodythat binds specifically to that marker and subsequently performing flowcytometric analysis of such a contacted cell to determine whether theantibody is bound the cell. In some embodiments, where agents specificfor cell-surface lineage markers used, the agents can all comprise thesame label or tag, such as fluorescent tag, and thus all cells positivefor that label or tag can be excluded or removed, to leave uncontactedhematopoietic stem or progenitor cells for use in the methods describedherein.

The term “lentivirus” refers to a group (or genus) of retroviruses thatgive rise to slowly developing disease. Viruses included within thisgroup include HIV (human immunodeficiency virus; including HIV type 1,and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes, i.e., T-cells.

The term “promoter/enhancer” refers to a segment of DNA which containssequences capable of providing both promoter and enhancer functions. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous,”“exogenous,” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

A “nucleic acid,” as described herein, can be RNA or DNA, and can besingle or double stranded, and can be selected, for example, from agroup including: nucleic acid encoding a protein of interest, forexample, transcription factors and reprogramming factors describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F collectively demonstrate the in vivo screening of TFsconfers functional hematopoiesis.

FIG. 1A. hPSC-derived HE was cultured for additional 3 days in EHTmedia, then infected with library of 26 TFs. Cells were injectedintrafemorally into sublethally irradiated (250 rads) NSG mice andtreated with doxycycline for 2 weeks to induce transgene expression.

FIG. 1B. Engraftment of human CD45+ cells was determined by flowcytometry of BM at 12 weeks. Library (N=6), 7 TFs (N=15), and 5 TFPoly(N=8). Defined 7 TFs and 5 TFPoly confer robust engraftment of HE. Thechimerism of human CD45+ population in BM was measured at 12 weeks. HEtransduced with either lentiviral library of HSC-specific TFs (Library);defined set of RUNX1, ERG, SPI1, LCOR, HOXA5, HOXA9, and HOXA10 (7 TFs);defined set of RUNX1, ERG, LCOR, HOXA5, and HOXA9 in polycistronicvectors (5 TFPoly).

FIG. 1C. Multilineage contribution of donor-derived cells in BM. After14 weeks, BM of NSG mice engrafted with TF library was analyzed formyeloid (M; CD33+), erythroid (E; GLY-A+), B-(CD19+), and T-cells (CD3+)within the human CD45+ population. Each bar represents individualrecipients engrafted. From left, recipient ID #1, #5 and #6 engraftedwith hiPSCs; recipient ID #2 left (L) femur and right (R) femur,recipient ID #3 left (L) femur and right (R) femur engrafted with hESCs;recipient ID #1 and #2 engrafted with CB-HSCs as a reference.

FIGS. 1D-1E. Primary transplantation of BM engrafted with 7 TF (FIG. 1D)and 5TFPoly (FIG. 1F) at 12 weeks. Human CD45+ BM of engrafted NSG wasanalyzed for HSCs (CD34+CD38−), nucleated erythroid (GLY-A+SYTO60+),enucleated erythroid (GLY-A+SYTO60−), neutrophils (PECAM+CD15+), B-cells(IgM+CD19+), B progenitor cells (IgM-CD19+), Plasmacytoid lymphocytes(IgM-CD19+CD38++) and T-cells (CD3+/CD4, CD8).

FIG. 1F. Lineage distribution of myeloid (CD33+), erythroid (GLY-A+),B-cells (CD19+) and T-cells (CD3+) was shown as a bar graph ofindividual recipients (N=5 for 7 TFs; N=4 for 5TFPoly).

FIGS. 2A-2G collectively demonstrate the detection of TFs that confermulti-lineage engraftment.

FIG. 2A. Transgene detection in engrafted cells of secondary recipientsof 7 TFs.

FIG. 2B. In vivo factor-minus-one (FMO) approach of defined 7 TFs toidentify any required or unnecessary factors. BM of NSG was analyzed at8 weeks for chimerism of human CD45+ population. The absence of RUNX1(0.33-fold, p=0.037), ERG (0.40-fold, p=0.056), LCOR (0.23-fold,p=0.020), HOXA5 (0.37-fold, p=0.056) or HOXA9 (0.26-fold, p=0.026)reduced chimerism. Lentiviral vector with GFP was used as negativecontrol. N=6 (4 for GFP). * p<0.05.

FIGS. 2C-2D. Secondary transplantation of BM engrafted with defined 7TFs. After 8 weeks from primary transplantation, 2,000 human CD34+ BMcells were transplanted to secondary recipients, and followed up to 8-14weeks. Compared with primary, secondary recipients had 0.34-fold fewerHSCs (p=0.12), 0.45-fold less nucleated erythroid (p=0.30), 3.3-foldmore enucleated erythroid (p=0.11), 1.6-fold more neutrophils (p=0.18),1.1-fold more mature B-cells (p=0.42), 0.65-fold more immature B-cells(p=0.10), and 0.56-fold less T cells (p=0.18). N=3 (primary), 2(secondary).

FIGS. 2E-2F. Phenotypic characterization of HSC-like cells in BMengrafted with 7 TF HSPCs. Human CD45+ population from BM of NSG miceengrafted with 7 TF HSPCs at 8 weeks was analyzed for human CD34 andCD38.

FIG. 2G. The population of HSCs (CD34+CD38−CD45+) was analyzed forcell-cycle state by Ki67 and DAPI. HSCs from NSG engrafted with CB-HSCsat the same time point were used as reference. N=3. The percentage ofHSCs in human CD45+ BM was 0.24-fold (p=0.058) (g); G0-phase was0.22-fold (p=0.0025); G1-phase was 2.3-fold (p=0.0052); S/G2/M was2.5-fold (p=0.013) vs CB equivalents (h). * p<0.01; ** p<0.05.

FIGS. 3A-3H collectively demonstrate the functional characterization ofterminally differentiated cells.

FIG. 3A. Globin switching in engrafted erythroid cells. Human GLY-A+cells were isolated from lysed BM (to exclude enucleated cells) of NSGengrafted with 7 TF HSPCs at 8 weeks and analyzed by qRT-PCR torelatively quantify HBE, HBG and HBB transcripts. Cytospin image ofisolated GLY-A+ cells are shown. CB; GLY-A+ erythroid cells fromCB-engrafted in NSG BM, 7 TF HSPCs; GLY-A+ erythroid cells from 7 TFHSPC-engrafted in NSG BM, 5F; GLY-A+ erythroid cells from hPSCstransduced with ERG, RORA, HOXA9, SOX4 and MYB17.

FIG. 3B. Enucleation of engrafted erythroid cells. BM of NSG miceengrafted with defined 7 TF HSPCs at 8 weeks time point was analyzed forhuman GLY-A and SYTO60. Cytospin images of RBCs from GLY-A+ populationsseparated by SYTO60 nuclear staining. Cells were isolated from unlysedBM of NSG engrafted with 7 TF HSPCs. Examples of nucleated andenucleated RBCs were shown. N=3.

FIG. 3C. Phenotyping of neutrophils. Human CD45+ population from BM ofNSG mice engrafted with defined 7 TF HSPCs at 8 weeks was analyzed forhuman PECAM and CD15. Myeloperoxidase activity of isolated CD45+PECAM+CD15+ neutrophils was measured with or without PMA stimulation.Neutrophils from NSG engrafted with CB-HSCs were used as reference. Thebasal level of MPO of HE was 0.40-fold less than CB (p=0.036). PMAstimulation increased MPO production 2.5-fold (p=0.010) (CB) and3.0-fold (p=0.10) (7 TF). Stimulated MPO production of 7 TF was0.47-fold (0.049) vs CB. * p<0.05. Data are from 2 independentexperiments with 3 technical replicates each time.

FIGS. 3D-3E. Measuring production of Ig in serum. Serum was isolatedfrom NSG mice engrafted with CB-HSCs or defined 7 TF HSPCs at 8 weeks(IgM) (FIG. 3D) and 14 weeks (IgG) (FIG. 3E). Production of IgM and IgG(ng/mL serum) was measured by ELISA. Serum from mock transplant and NSGengrafted with CB-HSCs was used as reference. The production of bothhuman IgM and IgG was detected and boosted by OVA in 7 TF HSPCs. *p<0.05. Data are from 2 independent experiments with 3 technicalreplicates each time.

FIG. 3F. Human CD3+ cells were isolated from BM of NSG mice engraftedwith CB-HSCs and defined 7 TF HSPCs at 8 weeks and cultured with orwithout PMA/lonomycin stimulation for 6 hours, when production of IFNγwas measured by ELISA. CD3+ T-cells from NSG engrafted with CB-HSCs wereused as reference. The basal level of IFNγ of HE was 0.53-fold (p=0.073)vs CB. PMA stimulation increased IFNγ production 4.4-fold (p=0.17) (CB)and 3.0-fold (p=0.16) (HE). Stimulated IFNγ production of HE was0.36-fold (0.039) vs CB. IFNγ production from CB-HSCs and HE themselveswere shown as reference. * p<0.05. Data are from 2 independentexperiments with 3 technical replicates each time.

FIG. 3G. Flow cytometric phenotyping of T-cells from engrafted 7 TFHSPCs. BM and thymus were collected at 8 weeks and analyzed for T cellmarkers (CD4, CD8, CD3, TCRαβ and TCRγδ). TCR phenotyping of the CD3+population was shown. One out of 3 recipients showed the presence ofTCRγδ. N=3.

FIG. 3H. TCR rearrangement of T-cells showed clonal diversity inhiPSC-derived cells. Human CD3+ thymocytes of NSG mice engrafted withdefined 7 TF HSPCs at 8 weeks was analyzed to detect TCR rearrangementby Immuno-seq. CD3+ Thymocytes from CB CD34+-engrafted NSG were used asa reference.

FIG. 4. Scheme of in vivo screening of transcription factors (TFs) toconfer functional hematopoiesis in hPSC-derived hemogenicendothelium(HE). Human ESCs/iPSCswere differentiated to embryoidbodies (EBs) bycytokines for specification of hemogenicendothelium (HE). At day 8 timepoint, CD34+FLK1+CD43-CD235A-HE was isolated, and cultured inendothelial-to-hematopoietic transition (EHT) media for additional 3days. Library of HSC-specific TFs was induced in HE via lentivirus andinjected to sub-lethally irradiated immunodeficientNSG miceintrafemoraly. Engrafted hematopoietic cells were isolated and analyzedby genomic PCR to detect integrated TFs (positive hits).

FIG. 5. Venn diagram of TFs that conferred in vivo engraftment and invitro CFU on PSC-HE.

DETAILED DESCRIPTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. It should beunderstood that this disclosure is not limited to the particularmethodology, protocols, and reagents, etc., described herein and as suchcan vary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe embodiments of the present disclosure, which is defined solely bythe claims.

Definitions of common terms in molecular biology can be found in TheMerck Manual of Diagnosis and Therapy, 19th Edition, published by MerckSharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), (2015 digital onlineedition at the website of Merck Manuals), Robert S. Porter et al.(eds.), The Encyclopedia of Molecular Cell Biology and MolecularMedicine, published by Blackwell Science Ltd., 1999-2012 (ISBN9783527600908); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Janeway's Immunobiology, KennethMurphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014(ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green andJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2012) (ISBN 1936113414); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS),John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and CurrentProtocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David HMargulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons,Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which areall incorporated by reference herein in their entireties. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Unless otherwise stated, the embodiments of the present disclosure wereperformed using standard procedures known to one skilled in the art, forexample, in Michael R. Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA (2012); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (1986);Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al.ed., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI)(John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), CurrentProtocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., JohnWiley and Sons, Inc.), Culture of Animal Cells: A Manual of BasicTechnique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005),Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P.Mather and David Barnes editors, Academic Press, 1st edition, 1998),Methods in Molecular biology, Vol. 180, Transgenesis Techniques by AlanR Clark editor, second edition, 2002, Humana Press, and Methods inMeolcular Biology, Vo. 203, 2003, Transgenic Mouse, editored by MartenH. Hofker and Jan van Deursen, which are all herein incorporated byreference in their entireties.

It should be understood that embodiments of the present disclosure arenot limited to the particular methodology, protocols, and reagents,etc., described herein and as such may vary. The terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the embodiments of the presentdisclosure, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages willmean±1%.

All patents and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the embodiments of the present disclosure. Thesepublications are provided solely for their disclosure prior to thefiling date of the present application. Nothing in this regard should beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior embodiments of the presentdisclosure or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The disclosure described herein, in a preferred embodiment, does notconcern a process for cloning human beings, processes for modifying thegerm line genetic identity of human beings, uses of human embryos forindustrial or commercial purposes or processes for modifying the geneticidentity of animals which are likely to cause them suffering without anysubstantial medical benefit to man or animal, and also animals resultingfrom such processes.

The disclosure described herein does not concern the destruction of ahuman embryo.

There is no one well tested and reliable method of producing HSCs andHSPCs from PSCs where these HSCs and HSPCs have all the hematopoieticlineage potentials and would engraft and reconstitute in vivo. i.e.,multi-lineage HSCs and HSPCs that capable of producing blood cells invivo. The disclosure seeks to provide improved PSCs-derived HSCs andHSPCs that exhibit long-term multilineage hematopoiesis in vivo afterimplantation in a host subject. For example, long-term multilineagehematopoiesis in vivo for at least 12 weeks or longer afterimplantation.

A variety of tissue lineages can be derived in vitro by stepwiseexposure of pluripotent stem cells (PSCs) to morphogens in an attempt tomimic embryonic development¹, or by conversion of one differentiatedcell type directly into another by enforced expression of mastertranscription factors (TFs)². Despite considerable effort, neitherapproach has yielded functional human hematopoietic stem cells (HSCs).Building upon recent evidence that HSCs derive from definitive hemogenicendothelium (HE)³⁻⁹, the inventors performed morphogen-directeddifferentiation of human PSCs into HE followed by combinatorialscreening of 26 candidate HSC-specifying TFs for the potential topromote hematopoietic engraftment in irradiated immune deficient murinehosts. The inventors recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10,LCOR, RUNX1, SPI1) that together were sufficient to convert HE intohematopoietic stem and progenitor cells (HSPCs) that engraft primary andsecondary murine recipients with myeloid cells, beta globin-expressingerythrocytes, IgM+/CD19+ B-cells, and αβ and γδ T-cells. Five TFs, ERG,HOXA5, HOXA9, LCOR, and RUNX1, are the minimum TFs necessary to convertHE into HSPCs. Integration analysis of virally-transduced transgenesdetected common clones in myeloid and lymphoid lineages, indicatingderivation of HSC-like cells from PSCs. This combined approach ofmorphogen-driven differentiation and TF-mediated cell fate conversionfrom PSCs yields HSPCs that hold promise for modeling hematopoieticdisease in humanized mice and for therapeutic strategies in geneticblood disorders.

Described herein, the inventors demonstrated a process of makingPSCs-derived HSCs and HSPCs that would differentiate into all thehematopoietic lineage potentials and would also engraft well in the hostafter transplantation so that there is sufficient engrafted cells tosustain blood production in vivo. This method produces functionallyrelevant HSCs and HSPCs in sufficient quantities for both meaningfulexperimental and therapeutic purposes. For example, in vitroexperiments, these PSCs-derived HSCs and HSPCs can be differentiated tothe desired hematopoietic lineage, e.g., erythroid cells, lymphoidcells, and myeloid cells, for further studies. For example, in in vivostudies, these PSCs-derived HSCs and HSPCs would engraft in the host,and differentiate into the variety of hematopoietic progeny cells, andreconstitute and populate the circulatory and immune system of the host.

Embodiments of the present disclosure are based, in part, to thediscovery of that transcription factors, ERG, HOXA5, HOXA9, LCOR, RUNX1,HOXA10, and SPI1, would bring about the differentiation of HSCs andHSPCs from PSC-derived hemogenic endothelia cells (HE). First, theinventors showed that embryonic bodies (EB) are made from pluripotentstem cells, e.g., including induced pluripotent cells. Second, the HEare harvested from the EB. Then, the HE cells are induced to undergo EHTand subsequently transfected with exogenous copies of at least thefollowing transcription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1, topromote differentiation of the HE into HSCs and HSPCs that exhibit allthe hematopoietic lineage potentials. These multi-lineage HSCs and HSPCsengraft in recipient host after implantation and made all kinds of bloodcells in vivo after implantation.

Accordingly, in one aspect, provided herein is a method for making HSCsand HSPCs comprising in vitro transfecting hemogenic endothelia cells(HE) with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs that engrafts in recipienthost after implantation.

In another aspect, this disclosure provides is a method of making HSCsand HSPCs comprising (a) generating EB from PSCs; (b) isolatinghemogenic endothelia cells (HE) from the resultant population of EB; (c)inducing EHT in culture in the isolated HE in order to obtainhematopoietic stem cells, and (d) in vitro transfecting the induced HEwith an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein thetranscription factors are expressed in the transfected cells to producea population of multilineage HSCs and HSPCs. In one embodiment, themethod further comprises selecting EBs that are generated from the PSCs,prior to isolating the HE. In one embodiment, the HE is isolated fromthe selected EBs.

In some aspects, this disclosure provides methods for enhancing orimproving the in vivo engraftment, or reconstitution, or both ofhematopoietic related cells that have been implanted into a subject. Inone embodiment, the method comprises providing populations ofmultilineage HSCs and HSPCs that have an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1, and optionally an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1, and implanting into a host subject. In oneembodiment, the multilineage HSCs and HSPCs further comprise and anexogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28. Inanother embodiment, the method comprises making HSCs and HSPCs by anyone method in this disclosure and implanting into a host subject. In oneembodiment, the subject donates some mature cells from which iPSCs areinduced. From the iPSCs, EBs are induced from which HEs are isolated andinduced to EHT, followed by the transfection of transcription factorgenes to produce HSCs and HSPCs. These HSCs and HSPCs are then implantedback into the donor subject, wherein the donor and recipient is thesubject. In one embodiment, a donor subject donates some mature cellsfrom which iPSCs are induced. From the iPSCs, EBs are induced from whichHEs are isolated and induced to EHT, followed by the transfection oftranscription factor genes to produce HSCs and HSPCs. These HSCs andHSPCs are then implanted back into a recipient subject, wherein thedonor and recipient are two different subjects.

In some aspects, this disclosure provides compositions of modified (alsoreferred to as engineered) cells for use in in vivo cellular replacementtherapy, for the manufacture of medicament for treatment ofhematological diseases, blood disorders, hematopoietic disorders, andfor in vitro studies of disease modeling, drug screening, andhematological diseases. In one embodiment, the engineered cells aremultilineage HSCs and HSPCs that have an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1, and optionally also contain an exogenous gene coding copy of thetranscription factors HOXA10, and SPI1. In one embodiment, theengineered cells are multilineage HSCs and HSPCs that further comprisean exogenous gene coding copy of each of the following reprogrammingfactors OCT4, SOX2, KLF4 and optionally c-MYC or NANOG and LIN28. Inanother embodiment, the engineered cells are HSCs and HSPCs made by anyone method described in this disclosure. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprising(a) generating EB from PSCs; (b) isolating HE from the resultantpopulation of EB; (c) inducing EHT in culture in the isolated HE inorder to obtain hematopoietic stem cells, and (d) in vitro transfectingthe population of HE with an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Inone embodiment, the method further comprises selecting EBs that aregenerated from the PSCs, prior to isolating the HE. In one embodiment,the HE is isolated from the selected EBs. In another embodiment, thepopulation of HE is further transfected with an exogenous gene codingcopy of the transcription factors HOXA10, and SPI1. In one embodiment,the engineered cells are the multilineage HSCs and HSPCs are producedfrom the resultant transfection of the described TFs. In one embodiment,the engineered cell further comprise an exogenous gene coding copy ofeach of the following reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or NANOG and LIN28. In one embodiment, the engineeredcells are CD34+. In another embodiment, the engineered cells are CD34+and CD45+. In another embodiment, the engineered cells are CD34+, CD45+,and CD38 negative.

In another aspect, this disclosure provides is an engineered cellderived from a population of HE that is produced by a method comprisingin vitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. In another embodiment, the population of HE is furthertransfected with an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1. In one embodiment, the engineered cells arethe multilineage HSCs and HSPCs are produced from the resultanttransfection of the described TFs. In one embodiment, the engineeredcell further comprise an exogenous gene coding copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC orNANOG and LIN28. In one embodiment, the engineered cells are CD34+. Inanother embodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is an engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. In another embodiment, theengineered cell further comprises an exogenous gene coding copy of thetranscription factors HOXA10, and SPI1. In one embodiment, theengineered cell further comprise an exogenous gene coding copy of eachof the following reprogramming factors OCT4, SOX2, KLF4 and optionallyc-MYC or NANOG and LIN28. In another embodiment, the engineered cellsare HSCs and HSPCs made by any one method described in this disclosure.In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising (a) generating embryonic bodies (EB)from pluripotent stem cells; (b) isolating hemogenic endothelia cells(HE) from the resultant population of EB; (c) inducing EHT in culture inthe isolated HE in order to obtain hematopoietic stem cells, and (d) invitro transfecting the population of HE with an exogenous gene codingcopy of each of the following transcription factors ERG, HOXA9, HOXA5,LCOR and RUNX1. In one embodiment, the population of HE is furthertransfected with an exogenous gene coding copy of the transcriptionfactors HOXA10, and SPI1. In one embodiment, the engineered cells arethe multilineage HSCs and HSPCs are produced from the resultanttransfection of the described TFs. In one embodiment, the engineeredcell further comprise an exogenous gene coding copy of each of thefollowing reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC orNANOG and LIN28. In one embodiment, the engineered cells are CD34+. Inanother embodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this composition is useful for cellular replacementtherapy in a subject.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells derived from a population of HE andproduced by a method comprising in vitro transfecting the population ofHE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In another embodiment, the engineered cells are HSCs and HSPCsmade by any one method described in this disclosure. In one embodiment,the engineered cells are CD34+. In another embodiment, the engineeredcells are CD34+ and CD45+. In another embodiment, the engineered cellsare CD34+, CD45+, and CD38 negative. In some embodiments, thiscomposition is useful for cellular replacement therapy in a subject, andfor in vitro studies of disease modeling, drug screening, andhematological diseases.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells wherein the cells comprise an exogenousgene coding copy of each of the following transcription factors ERG,HOXA9, HOXA5, LCOR and RUNX1. In one embodiment, the cells furthercomprise an exogenous gene coding copy of the transcription factorsHOXA10, and SPI1. In another embodiment, the engineered cells are HSCsand HSPCs made by any one method described in this disclosure. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising (a) generating EBfrom PSCs; (b) isolating HE from the resultant population of EB; (c)inducing EHT in culture in the isolated HE in order to obtainhematopoietic stem cells, and (d) in vitro transfecting the populationof HE with an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this pharmaceutical composition is useful for cellularreplacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells derived from apopulation of HE and a pharmaceutically acceptable carrier, wherein theengineered cells are produced by a method comprising in vitrotransfecting the population of HE with an exogenous gene coding copy ofeach of the following transcription factors ERG, HOXA9, HOXA5, LCOR andRUNX1. In one embodiment, the population of HE is further transfectedwith an exogenous gene coding copy of the transcription factors HOXA10,and SPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative. Insome embodiments, this pharmaceutical composition is useful for cellularreplacement therapy in a subject.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells and apharmaceutically acceptable carrier, wherein the engineered cellscomprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the engineered cells further comprise an exogenous genecoding copy of the transcription factors HOXA10, and SPI1. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells are produced by a method comprising (a) generating EB from PSCs;(b) isolating HE from the resultant population of EB; (c) inducing ENTin culture in the isolated HE in order to obtain hematopoietic stemcells, and (d) in vitro transfecting the population of HE with anexogenous gene coding copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1. In one embodiment, thepopulation of HE is further transfected with an exogenous gene codingcopy of the transcription factors HOXA10, and SPI1. In one embodiment,the engineered cells are CD34+. In another embodiment, the engineeredcells are CD34+ and CD45+. In another embodiment, the engineered cellsare CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells are produced by a method comprising in vitro transfecting thepopulation of HE with an exogenous gene coding copy of each of thefollowing transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. Inone embodiment, the population of HE is further transfected with anexogenous gene coding copy of the transcription factors HOXA10, andSPI1. In one embodiment, the engineered cells are CD34+. In anotherembodiment, the engineered cells are CD34+ and CD45+. In anotherembodiment, the engineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells to a recipient subject, the population of engineeredcells comprise an exogenous gene coding copy of each of the followingtranscription factors ERG, HOXA9, HOXA5, LCOR and RUNX1. In oneembodiment, the engineered cells further comprise an exogenous genecoding copy of the transcription factors HOXA10, and SPI1. In oneembodiment, the engineered cells are CD34+. In another embodiment, theengineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides engineered cells derivedfrom a population of HE and produced by a method described herein. Inone embodiment, the engineered cells are CD34+. In another embodiment,the engineered cells are CD34+ and CD45+. In another embodiment, theengineered cells are CD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a composition comprisinga population of engineered cells described herein. In one embodiment,the engineered cells are multilineage HSCs and HSPCs are produced fromthe resultant transfection of the described TFs. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition comprising a population of engineered cells described hereinand a pharmaceutically acceptable carrier. In one embodiment, theengineered cells are CD34+. In another embodiment, the engineered cellsare CD34+ and CD45+. In another embodiment, the engineered cells areCD34+, CD45+, and CD38 negative.

In another aspect, this disclosure provides is a pharmaceuticalcomposition described herein for use in cellular replacement therapy ina subject.

In another aspect, this disclosure provides is a method of cellularreplacement therapy in a subject in need thereof, the method comprisingadministering a therapeutically effective amount of a population ofengineered cells described, or a composition described, or apharmaceutical composition described to a recipient subject.

In one embodiment of any one aspect described, the method of generatingHSCs and HSPCs described is an in vitro or ex vivo method.

In one embodiment of any one method, engineered cell, or compositiondescribed, the multilineage hematopoietic progenitor cells are generatedby introducing in vitro or ex vivo each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1, in the HE cells derived fromPSC-induced EBs. For example, by transfecting with a vector or more, thevector(s) collectively carry an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for in vivo expression of the transcription factor in the transfectedcells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the multilineage hematopoietic progenitor cells are generatedby contacting a population of HEs with a vector or more, wherein thevector(s) collectively carrying an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for the in vivo expression of the factors in the contacted cells, andwherein the transfected transcription factors are expressed in vivo inthe contacted cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factor, HOXA10,wherein the transfected transcription factor is expressed in vivo in thetransfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factor, SPI1,wherein the transfected transcription factor is expressed in vivo in thetransfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the method further comprising in vitro transfecting the HEwith an exogenous gene coding copy of the transcription factors, HOXA10and SPI1, wherein the transfected transcription factors are expressed invivo in the transfected cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell of this disclosure is a mammalian cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered mammalian cell is a primate cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered primate cell is a human cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+ and CD45+.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell is CD34+, CD45+, and CD38 negative.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell has an exogenous gene of at least one ofthe following transcription factors: ERG, HOXA9, HOXA5, HEXA10, SPI1,LCOR and RUNX1.

In another embodiment of any one method, engineered cell, or compositiondescribed, the engineered cell has an exogenous gene of at least one ofthe following reprogramming factors: OCT4, SOX2, KLF4, c-MYC, NANOG, andLIN28.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that engraft in vivo in a host recipient subject and produceblood cells in vivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that reconstitutes the hematopoietic system in vivo whentransplanted into a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that differentiate to myeloid cells in vivo, and the myeloidcells produce MPO upon PMA or cytokine stimulation in vivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells disclosed herein are multilineage HSCsand HSPCs that differentiate to functional T- and B-cells in vivo, thefunctional T- and B-cells produce IgM and IgG. The functional T- andB-cells also undergo immunoglobulin class switching in response toovalbumin stimulation. The functional T- and B-cells also producesINF-γ.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells produce blood cells in vivo whenengrafted in vivo in a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells reconstitutes the hematopoietic systemin vivo when transplanted into a host recipient subject.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells differentiate to myeloid cells in vivo,and the myeloid cells produce MPO upon PMA or cytokine stimulation invivo.

In one embodiment of any one method, engineered cell, or compositiondescribed, the engineered cells differentiate to functional immune cellsin vivo, e.g., T- and B-cells, wherein the functional immune cellsproduce IgM and IgG. The functional immune cells also undergoimmunoglobulin class switching in response to ovalbumin stimulation. Thefunctional immune cells also produce INF-γ.

In one embodiment of any one method, engineered cell, or compositiondescribed, the HE cells are cells that are derived from embryoid bodies(EBs) obtained from a population of pluripotent stem cells (PSC). In oneembodiment, the HE are definitive HE.

In one embodiment of any one method, engineered cell, or compositiondescribed, the population of PSC is induced pluripotent stem cells(iPSc) or embryonic stem cells (ESC).

In one embodiment of any one method, engineered cell, or compositiondescribed, the PSC are human PSC or mouse PSC.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSCs are produced by in vitro or ex vivo introducingexogenous copies of only three reprogramming factors OCT4, SOX2, andKLF4 into mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC having exogenous copies of OCT4, SOX2, and KLF4 isfurther introduced in vitro or ex vivo with an exogenous copy of c-MYCinto the cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC having exogenous copies of OCT4, SOX2, and KLF4 isfurther introduced in vitro or ex vivo with an exogenous copies of NANOGand LIN28 into the cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by introducing in vitro or ex vivoexogenous copies of reprogramming factors OCT4, SOX2, and KLF4, andoptionally with c-MYC or NANOG and LIN28 into the mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo contacting themature cells with a vector or more, wherein the vector(s) collectivelycarry exogenous copies of reprogramming factors OCT4, SOX2, and KLF4,and optionally with c-MYC or NANOG and LIN28 into mature cells, andwherein the reprogramming factors are expressed in vivo in the contactedmature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made can be from anycell type in a donor subject. For examples, cells from a blood sample,or bone marrow sample, B lymphocytes (B-cells), T lymphocytes,(T-cells), fibroblasts, keratinocytes etc. In some embodiments, anymature cell type from the donor subject can be used except a cell withno nucleus, such as a human red blood cell.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo introducing thedisclosed reprogramming factors two or more times into the mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the iPSC are produced by in vitro or ex vivo contacting themature cells with the disclosed vector(s) factors two or more times intothe mature cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are mammaliancells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are primate cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are human cells.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are autologous to arecipient subject who would be receiving the engineered cells that arederived from the mature cells according to any one of the methodsdescribed in this disclosure.

In one embodiment of any one method, engineered cell, or compositiondescribed, the mature cells from which iPSC are made are HLA matchedwith a recipient subject who would be receiving the engineered cellsthat are derived from the mature cells according to any one of themethods described in this disclosure.

In one embodiment of any one engineered cell or composition described,the pharmacological acceptable carrier is not cell culture media.

In one embodiment of any one composition described, the pharmacologicalcomposition is a cryopreserved composition comprising at least onecryopreservative agent known in the art. For examples, dimethylsulphoxide (DMSO), polyvinylpyrrolidone (PVP), fetal calf serum (FCS),polyethylene glycol (PEG), glycerol, ethylene glycol (EG) and trehalose.Combinations of two or more cryopreservative agents can be used.

Pluripotent Stem Cells for Generating Embryonic Bodies, HE, and HSC andHSPC.

Pluripotent stem cells (PSCs) have the potential to give rise to all thesomatic tissues. Directed differentiation of PSCs aims to recapitulateembryonic development to generate patient-matched tissues by specifyingthe three germ layers. A common theme in directed differentiation acrossall germ layers is the propensity of PSCs to give rise to embryonic- andfetal-like cell types, which poses a problem for integration andfunction in an adult recipient. This distinction is particularlystriking in the hematopoietic system, which emerges in temporally andspatially separated waves at during ontogeny (Dzierzak and Speck, 2008).The earliest “primitive” progenitors emerge in the yolk sac at 8.5 dpcand give rise to a limited repertoire of macrophages, megakaryocytes andnucleat-ed erythrocytes (Baron et al 2005, Tavian and Peault 2005,Ferkowicz et al 2005). These early embryonic-like progenitors aregenerally myeloid-based and cannot functionally repopulate the bonemarrow of adult recipients. By contrast, “definitive” cells withhematopoietic stem cell (HSC) potential emerge later in arterialendothelium within the aorta-gonad-mesonephros (AGM) and otheranatomical sites (Dzierzak and Speck, 2008). Directed differentiation ofPSCs gives rise to hematopoietic progenitors, which resemble those foundin the yolk sac of the early embryo. These lack functionalreconstitution potential, are biased to myeloid lineages, and expressembryonic globins.

In one embodiment of any one aspect described, the population of PSCused for generating EBs is induced pluripotent stem cells (iPS cells) orembryonic stem cells (ESC).

In one embodiment of any one aspect described, the iPS cells areproduced by introducing only reprogramming factors OCT4, SOX2, KLF4 andoptionally c-MYC or NANOG and LIN28 into mature cells.

In one embodiment of any one aspect described, the mature cells forproducing iPS cells are selected from the group consisting of Blymphocytes (B-cells), T lymphocytes, (T-cells), fibroblasts, andkeratinocytes.

In one embodiment of any one aspect described, the iPSCs are produced byintroducing the reprogramming factors two or more times into the maturecells.

Induced Pluripotent Stem Cells

In some embodiments, the pluripotent stem cells (PSCs) described hereinare derived from isolated induced pluripotent stem cells (iPSCs). Anadvantage of using iPSCs is that the cells can be derived from the samesubject to which the eventual immune cells would be reintroduced. Thatis, a somatic cell can be obtained from a subject, reprogrammed to aninduced pluripotent stem cell, and then transfected and differentiatedinto a modified immune cell to be administered to the subject (e.g.,autologous cells). Since the progenitors are essentially derived from anautologous source, the risk of engraftment rejection or allergicresponses is reduced compared to the use of cells from another subjector group of subjects. In some embodiments, the cells for generatingiPSCs are derived from non-autologous sources. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one embodiment, the PSCs used in the disclosed methods are notembryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to induced pluripotent stem cells. Exemplary methods areknown to those of skill in the art and are described briefly hereinbelow.

As used herein, the term “reprogramming” refers to a process that altersor reverses the differentiation state of a differentiated cell (e.g., asomatic cell). Stated another way, reprogramming refers to a process ofdriving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cellsincluded in the term differentiated cells does not render these cellsnon-differentiated cells (e.g., undifferentiated cells) or pluripotentcells. The transition of a differentiated cell to pluripotency requiresa reprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. In some embodiments,reprogramming encompasses complete reversion of the differentiationstate of a differentiated cell (e.g., a somatic cell) to a pluripotentstate or a multipotent state. In some embodiments, reprogrammingencompasses complete or partial reversion of the differentiation stateof a differentiated cell (e.g., a somatic cell) to an undifferentiatedcell (e.g., an embryonic-like cell). Reprogramming can result inexpression of particular genes by the cells, the expression of whichfurther contributes to reprogramming. In certain embodiments describedherein, reprogramming of a differentiated cell (e.g., a somatic cell)causes the differentiated cell to assume an undifferentiated state(e.g., is an undifferentiated cell). The resulting cells are referred toas “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs oriPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, and epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a common myeloid stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some embodiments.

The specific approach or method used to generate pluripotent stem cellsfrom somatic cells (broadly referred to as “reprogramming”) is notcritical to the claimed embodiments of the present disclosure. Thus, anymethod that re-programs a somatic cell to the pluripotent phenotypewould be appropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described toinduced pluripotent stem cells from somatic cells. Yamanaka andTakahashi converted mouse somatic cells to ES cell-like cells withexpanded developmental potential by the direct transduction of Oct4,Sox2, Klf4, and optionally c-Myc. See U.S. Pat. Nos. 8,058,065 and9,045,738 to Yamanaka and Takahashi. iPSCs resemble ES cells as theyrestore the pluripotency-associated transcriptional circuitry and muchof the epigenetic landscape. In addition, mouse iPSCs satisfy all thestandard assays for pluripotency: specifically, in vitro differentiationinto cell types of the three germ layers, teratoma formation,contribution to chimeras, germline transmission, and tetraploidcomplementation.

Subsequent studies have shown that human iPS cells can be obtained usingsimilar transduction methods, and the transcription factor trio, OCT4,SOX2, and NANOG, has been established as the core set of transcriptionfactors that govern pluripotency. The production of iPS cells can beachieved by the introduction of nucleic acid sequences encoding stemcell-associated genes into an adult, somatic cell, using viral vectors.In general, retroviruse- or adenoviruse-based vectors are used todeliver the desired gene/nucleic acid. Other viruses used as vectorsinclude adeno-associated viruses, lentiviruses, pox viruses,alphaviruses, and herpes viruses.

iPS cells can be generated or derived from terminally differentiatedsomatic cells, as well as from adult stem cells, or somatic stem cells.That is, a non-pluripotent progenitor cell can be rendered pluripotentor multipotent by reprogramming. In such instances, it may not benecessary to include as many reprogramming factors as required toreprogram a terminally differentiated cell. Further, reprogramming canbe induced by the non-viral introduction of reprogramming factors, e.g.,by introducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5):618-30, this referenceis incorporated herein by reference in its entirety.). Reprogramming canbe achieved by introducing a combination of nucleic acids encoding stemcell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can further comprise introducing one or more of Oct-3/4, a memberof the Sox family, a member of the Klf family, and a member of the Mycfamily to a somatic cell. In one embodiment, the methods andcompositions described herein further comprise introducing one or moreof each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. Asnoted above, the exact method used for reprogramming is not necessarilycritical to the methods and compositions described herein. However,where cells differentiated from the reprogrammed cells are to be usedin, e.g., human therapy, in one embodiment the reprogramming is noteffected by a method that alters the genome. Thus, in such embodiments,reprogramming is achieved, e.g., without the use of viral or plasmidvectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various small molecules as shown by Shi, Y., et al (2008)Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135.This reference is incorporated herein by reference in its entirety.Thus, an agent or combination of agents that enhance the efficiency orrate of induced pluripotent stem cell production can be used in theproduction of patient-specific or disease-specific iPSCs. Somenon-limiting examples of agents that enhance reprogramming efficiencyinclude soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histonemethyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferaseinhibitors, histone deacetylase (HDAC) inhibitors, valproic acid,5′-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA),vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherreprogramming enhancing agents include, for example, dominant negativeforms of the HDACs (e.g., catalytically inactive forms), siRNAinhibitors of the HDACs, and antibodies that specifically bind to theHDACs. Such inhibitors are available, e.g., from BIOMOL International,Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, AtonPharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, andSigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection does not involve only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, while cell surface markers are readilyidentified, e.g., by immunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate to cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced to nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Many US Patents and Patent Application Publications teach and describemethods of generating iPSCs and related subject matter. For examples,U.S. Pat. Nos. 9,347,044, 9,347,042, 9,347,045, 9,340,775, 9,341,625,9,340,772, 9,250,230, 9,132,152, 9,045,738, 9,005,975, 9,005,976,8,927,277, 8,993,329, 8,900,871, 8,852,941, 8,802,438, 8,691,574,8,735,150, 8,765,470, 8,058,065, 8,048,675, and US Patent PublicationNos: 20090227032, 20100210014, 20110250692, 20110201110, 20110200568,20110306516, 20100021437, 20110256626, 20110044961, 20120276070,20120263689, 20120128655, 20120100568, 20130295064, 20130029866,20130189786, 20130295579, 20130130387, 20130157365, 20140234973,20140227736, 20140093486, 20140301988, 20140170746, 20140178989,20140349401, 20140065227, and 20150140662. These references areincorporated herein by reference in their entirety.

Somatic Cells for Reprogramming

Somatic cells, as that term is used herein, refer to any cells formingthe body of an organism, excluding germline cells. Every cell type inthe mammalian body—apart from the sperm and ova, the cells from whichthey are made (gametocytes) and undifferentiated stem cells—is adifferentiated somatic cell. For example, internal organs, skin, bones,blood, and connective tissue are all made up of differentiated somaticcells.

Additional somatic cell types for use with the compositions and methodsdescribed herein include: a fibroblast (e.g., a primary fibroblast), amuscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammarycell, an hepatocyte and a pancreatic islet cell. In some embodiments,the somatic cell is a primary cell line or is the progeny of a primaryor secondary cell line. In some embodiments, the somatic cell isobtained from a human sample, e.g., a hair follicle, a blood sample, abiopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g.,an oral swab sample), and is thus a human somatic cell.

Some non-limiting examples of differentiated somatic cells include, butare not limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, skin, immune cells, hepatic, splenic, lung, peripheralcirculating blood cells, gastrointestinal, renal, bone marrow, andpancreatic cells. In some embodiments, a somatic cell can be a primarycell isolated from any somatic tissue including, but not limited tobrain, liver, gut, stomach, intestine, fat, muscle, uterus, skin,spleen, endocrine organ, bone, etc. Further, the somatic cell can befrom any mammalian species, with non-limiting examples including amurine, bovine, simian, porcine, equine, ovine, or human cell. In someembodiments, the somatic cell is a human somatic cell.

When reprogrammed cells are used for generation of thyroid progenitorcells to be used in the therapeutic treatment of disease, it isdesirable, but not required, to use somatic cells isolated from thepatient being treated. For example, somatic cells involved in diseases,and somatic cells participating in therapeutic treatment of diseases andthe like can be used. In some embodiments, a method for selecting thereprogrammed cells from a heterogeneous population comprisingreprogrammed cells and somatic cells they were derived or generated fromcan be performed by any known means. For example, a drug resistance geneor the like, such as a selectable marker gene can be used to isolate thereprogrammed cells using the selectable marker as an index.

Reprogrammed somatic cells as disclosed herein can express any number ofpluripotent cell markers, including: alkaline phosphatase (AP); ABCG2;stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60;TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-III-tubulin; α-smoothmuscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Daxl;zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cellassociated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7;ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fth117; Sal14;undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53;G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a;Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4;Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3;CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-celllymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markersfor pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3;Grb2; β-catenin, and Bmi1. Such cells can also be characterized by thedown-regulation of markers characteristic of the somatic cell from whichthe induced pluripotent stem cell is derived.

Embryonic Bodies (EBs) from Pluripotent Stem Cells

In one embodiment of any one aspect described, the EB are generated orinduced from PSCs, for example, iPSCs or embryonic stem cells (ESCs)derived from the blastocyst stage of embryos from mouse (mESC), primate,and human (hESC) sources.

EBs are three-dimensional aggregates of pluripotent stem cells producedand cultured in vitro in the presence of serum. EBs largely exhibitheterogeneous patterns of differentiated cell types, generating amixture of primitive and definitive hematopoietic progenitor cell types.Primitive progenitors equate to those that arise in vivo naturally inthe earliest stages of embryonic development, whereas at later stages ofmaturation the embryonic populations give rise to definitive progenitorscells, which behave similarly to the cells typical of adulthematopoiesis.

EB formation is often used as a method for initiating spontaneousdifferentiation toward the three germ lineages. EB differentiationbegins with the specification of the exterior cells toward the primitiveendoderm phenotype. The cells at the exterior then deposit extracellularmatrix (ECM), containing collagen IV and laminin, similar to thecomposition and structure of basement membrane. In response to the ECMdeposition, EBs often form a cystic cavity, whereby the cells in contactwith the basement membrane remain viable and those at the interiorundergo apoptosis, resulting in a fluid-filled cavity surrounded bycells. Subsequent differentiation proceeds to form derivatives of thethree germ lineages. In the absence of supplements, the “default”differentiation of ESCs is largely toward ectoderm, and subsequentneural lineages. However, alternative media compositions, including theuse of fetal bovine serum as well as defined growth factor additives,have been developed to promote the differentiation toward mesoderm andendoderm lineages.

As a result of the three-dimensional EB structure, complex morphogenesisoccurs during EB differentiation, including the appearance of bothepithelial- and mesenchymal-like cell populations, as well as theappearance of markers associated with the epithelial-mesenchymaltransition (EMT). Additionally, the inductive effects resulting fromsignaling between cell populations in EBs results in spatially andtemporally defined changes, which promote complex morphogenesis.Tissue-like structures are often exhibited within EBs, including theappearance of blood islands reminiscent of early blood vessel structuresin the developing embryo, as well as the patterning of neuriteextensions (indicative of neuron organization) and spontaneouscontractile activity (indicative of cardiomyocyte differentiation) whenEBs are plated onto adhesive substrates such as gelatin. More recently,complex structures, including optic cup-like structures were created invitro resulting from EB differentiation.

A non-limited method for producing EBs from iPSC is as follows. HumaniPSCs were differentiated as EBs in the presence of BMP4 and cytokines,as previously described (Chadwick et al., 2003, Blood, 102:906-915).Briefly, iPSC colonies were scraped into non-adherent rotating 10 cmplates. EB media was KO-DMEM+20% FBS (Stem Cell Technologies), 1 mML-glutamine, 1 mM NEAA, penicillin/streptomycin, 0.1 mMβ-mercaptoethanol, 200 μg/ml h-transferrin, and 50 μg/ml ascorbic acid.After 24 hrs, media was changed by allowing EBs to settle by gravity,and replaced with EB media supplemented with growth factors: 50 ng/mlBMP4 (R&D Systems), 300 ng/ml SCF, 300 ng/ml FLT3, 50 ng/ml G-CSF, 20ng/ml IL-6, 10 ng/ml IL-3 (all Peprotech). Media was changed on day 3,5, 8 and 10. EBs were observed by day 5. EBs can be selected anddissociated to individual cells anywhere from day 8-14 by digesting withcollagenase B (Roche) for 2 hrs, followed by treatment with enzyme-freedissociation buffer (Gibco), and filtered through an 80 μm filter.

An alternative method of producing EBs from iPSC is as follows.Confluent human or mouse iPSC colonies were disrupted to smallaggregates using collagenase IV (1 mg/ml, 15 minutes at 37° C.). Cellaggregates were resuspended in StemPro fully defined medium (StemPro-34Serum-Free Media, Gibco proprietary formulation, guaranteed as animalprotein-free by the manufacturer) supplemented with 0.5 ng/ml humanrecombinant BMP-4 (R&D Systems) at a ratio of 2 confluent ESC/iPSC wellsfor 1 well of differentiation. During all the differentiation process,cell attachment was prevented using low cluster tissue culture dishes(Corning Costar). On day 1 of differentiation, embryoid bodies (EB) wereharvested and transferred to fresh StemPro media supplemented with 10ng/ml BMP-4 and 5 ng/ml bFGF. After 72 hours, EB were harvested againand transferred to a medium consisting of StemPro media supplementedwith 100 ng/ml human recombinant VEGF (R&D Systems), 5 ng/ml bFGF, 100ng/ml SCF (kind gift from Amgen), 100 ng/ml FLT3-L (kind gift fromAmgen) and 40 ng/ml TPO (Peprotech, UK) for 4 additional days. Alldifferentiation steps were performed in hypoxic conditions (5% O₂) in ahumidified incubator at 37° C. After 8 days of differentiation, EB wereharvested and spun at 65 g for 4 min. Supernatants containingnon-adherent cells were stored on ice and EB were disrupted usingTrypsin-EDTA followed by manual disruption in FBS containing blockingmedium using a 21G needle, After centrifugation, cells were resuspendedin freshly prepared collagenase IV solution (0.2 mg/ml) for 30 min at37° C. Cells were disrupted again using a 21G needle and filtered over a70 μm cell strainer (Falcon). Supernatants from the initialcentrifugation were pooled with these suspensions and magnetic beadassociated cell sorting (MACS) for the CD34 epitope was performedfollowing manufacturer's instructions (Miltenyi Biotech).

Other methods of generating EBs from ESC and iPSC are known in the art.For example, as described in U.S. Pat. Nos. 6,602,711; 7,220,584;7,452,718; 7,648,833; 7,795,026; 7,803,619; 8,278,097, 8,501,474; and8,986,996, the contents of each are incorporated herein by reference inits entirety.

In one embodiment of any one aspect described, the EB are generated orinduced from PSCs by culturing or exposing the PSCs to morphogens forabout 8 days.

In one embodiment of any one aspect described, the exposure to themorphogens is for about 5-14 days, 5-13 days, 5-12 days, 5-11 days, 5-10days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 6-14 days, 6-13 days, 6-12days, 6-11 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-14 days,7-13 days, 7-12 days, 7-11 days, 7-10 days, 7-9 days, 7-8 days, 8-14days, 8-13 days, 8-12 days, 8-11 days, 8-10 days, 8-9 days, 9-14 days,9-13 days, 9-12 days, 9-11 days, 9-10 days, 10-14 days, 10-13 days,10-12 days, 10-11 days, 11-14 days, 11-13 days, 11-12 days, 12-14 days,12-13 days, or 13-14 days.

In one embodiment of any one aspect described, the exposure to themorphogens of the PSCs is for about 5 days, 6 days, 7 days, 9 days, 10days, 11 days 12 days, 13 days or 14 days.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs is/are selected from the groupconsisting of holo-transferrin, mono-thioglycerol (MTG), ascorbic acid,bone morphogenetic protein (BMP)-4, basic fibroblast growth factor(bFGF), SB431542, CHIR99021, vascular endothelial growth factor (VEGF),interleukin (IL)-6, insulin-like growth factor (IGF)-1, interleukin(IL)-11, stem cell factor (SCF), erythropoietin (EPO), thrombopoietin(TPO), interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand(Flt-3L).

In some embodiments of any one aspect described, a combination of two ormore of these morphogens are selected to generate EBs from PSCs.

In one embodiment of any one aspect described, a combination of allmorphogens holo-transferrin, MTG, ascorbic acid, BMP-4, bFGF, SB431542,CHIR99021, VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, IL-3, and Flt-3L areused to generate EBs from PSCs.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs consist essentially ofholo-transferrin, MTG, ascorbic acid, BMP-4, bFGF, SB431542, CHIR99021,VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, IL-3, and Flt-3L.

In one embodiment of any one aspect described, the morphogens forinducing EBs formation from PSCs consist of holo-transferrin, MTG,ascorbic acid, BMP-4, bFGF, SB431542, CHIR99021, VEGF, IL-6, IGF-1,IL-11, SCF, EPO, TPO, IL-3, and Flt-3L.

In one embodiment of any one aspect described, the method furthercomprises selecting EBs that are generated from the PSCs, prior toisolating HE from the selected EBs.

In one embodiment of any one aspect described, the desired EBs are lessthan 800 microns in size and are selected.

In other embodiments of any one aspect described, the EBs selected areless than 790 μm, less than 780 μm, less than 770 μm, less than 760 μm,less than 750 μm, less than 740 μm, less than 730 μm, less than 720 μm,less than 710 μm, less than 700 μm, less than 690 μm, less than 680 μm,less than 670 μm, less than 660 μm, less than 650 μm, less than 640 μm,less than 630 μm, less than 620 μm, less than 610 μm, less than 600 μm,less than 590 μm, less than 580 μm, less than 570 μm, less than 560 μm,less than 550 μm, less than 540 μm, less than 530 μm, less than 520 μm,less than 510 μm, less than 500 μm, less than 490 μm, less than 480 μm,less than 470 μm, less than 460 μm, less than 450 μm, less than 440 μm,less than 430 μm, less than 420 μm, less than 410 μm, less than 400 μm,less than 390 μm, less than 380 μm, less than 370 μm, less than 360 μm,less than 350 μm, less than 340 μm, less than 330 μm, less than 320 μm,less than 310 μm, less than 300 μm, less than 290 μm, less than 280 μm,less than 270 μm, less than 260 μm, less than 250 μm, less than 240 μm,less than 230 μm, less than 220 μm, less than 210 μm, less than 200 μm,less than 190 μm, less than 180 μm, less than 170 μm, less than 160 μm,less than 150 μm, less than 140 μm, less than 130 μm, less than 120 μm,less than 110 μm, or less than 100 μm in size.

In other embodiments of any one aspect described, the EBs selected areabout 800 μm, about 790 μm, about 780 μm, about 770 μm, about 760 μm,about 750 μm, about 740 μm, about 730 μm, about 720 μm, about 710 μm,about 700 μm, about 690 μm, about 680 μm, about 670 μm, about 660 μm,about 650 μm, about 640 μm, about 630 μm, about 620 μm, about 610 μm,about 600 μm, about 590 μm, about 580 μm, about 570 μm, about 560 μm,about 550 μm, about 540 μm, about 530 μm, about 520 μm, about 510 μm,about 500 μm, about 490 μm, about 480 μm, about 470 μm, about 460 μm,about 450 μm, about 440 μm, about 430 μm, about 420 μm, about 410 μm,about 400 μm, about 390 μm, about 380 μm, about 370 μm, about 360 μm,about 350 μm, about 340 μm, about 330 μm, about 320 μm, about 310 μm,about 300 μm, about 290 μm, about 280 μm, about 270 μm, about 260 μm,about 250 μm, about 240 μm, about 230 μm, about 220 μm, about 210 μm,about 200 μm, about 190 μm, about 180 μm, about 170 μm, about 160 μm,about 150 μm, about 140 μm, about 130 μm, about 120 μm, about 110 μm, orabout 100 μm in size.

The EB size selection can be achieved by any method known in the art.For example, by filtration through a filter with the desired pore size,e.g., a 200 μm filter. For example, selection is done visually.

In one embodiment of any one aspect described, the EB cells within theEBs selected are compactly adhered to each other and are dissociated toindividual EB cells.

In one embodiment of any one aspect described, the EB cells within theEBs selected are dissociated to individual EB cells by digestion withtrypsin or collagenase.

In one embodiment of any one aspect described, the EB cells of theselected EBs are dissociated to individual EB cells prior to theisolation of HE.

Hemogenic Endothelia Cells (HE) Isolated from the Dissociated IndividualEB Cells

Hemogenic endothelium (HE) is a special subset of endothelial cellsscattered within blood vessels that can differentiate into hematopoieticcells. During embryonic development, multilineage HSCs/progenitor cellsare derived from specialized endothelial cells, termed hemogenicendothelium, within the yolk sac, placenta, and aorta. The multilineageHSCs/progenitor cells responsible for the generation of all blood celltypes during definitive hematopoiesis arise from hemogenic endothelium.All blood cells emerge from hemogenic endothelial-expressing cellsthrough an endothelial-to-hematopoietic transition (EHT).

In vitro differentiation EB produces a hetergenous population of cells,including cells that are hemogenic endothelial-like. These are the HEcells that are isolated and induced to under go EHT in culture. In oneembodiment of any one aspect described, the HE are isolated from theselected and dissociated EB cells.

In one embodiment of any one aspect described, the HE are isolatedimmediately from the selected and dissociated EB cells. In otherembodiments of any one aspect described, the HEs are isolated less thanthree hours, less than two hours, less than one hour, less than 55 min,less than 50 min, less than 45 min, less than 40 min, less than 35 min,less than 30 min, less than 25 min, less than 20 min, less than 15 min,and less than 10 min after the dissociation of the select EBs toindividual EB cells. In other embodiments of any one aspect described,the HEs are isolated within three hours, within two hours, within onehour, within 55 min, within 50 min, within 45 min, within 40 min, within35 min, within 30 min, within 25 min, within 20 min, within 15 min, andwithin 10 min after the dissociation of the select EBs to individual EBcells.

In one embodiment of any one aspect described, the isolated HE aredefinitive HE. Definitive HE is a population that is defined bycombination of surface antigen markers CD34+FLK1+CD235A−CD43−.Definitive hematopoietic stem cells (HSCs) are the cells responsible forthe continuous production of all mature blood cells during the entireadult life span of an individual. Similarly, definitive HE are the cellsthat are responsible for the continuous production of all mature bloodcells during the entire adult life span of an individual.

In one embodiment of any one aspect described, the isolated HE areFLK1+, CD34+, CD43− and CD235A−.

In one embodiment of any one aspect described, the isolated HE areFlk1+cKit+CD45−.

These are the cell surface biomarkers on the isolated HE before theendothelial-to-hematopoietic transition (EHT).

The HEs can be isolated by any method known in the art. For example, byimmune-magnetic beads directed to the cell surface biomarkers that arecharacteristics of HEs. For example, by positive selection for FLK1+ andCD34+ cells from the dissociated individual EB cells followed bynegative selection for CD43+ cells, and for CD235A+ cells from theresultant FLK1+ and CD34+ cells. FLK1+ and CD34+ cells that are CD43+are discarded. FLK1+ and CD34+ cells that are CD235A+ are alsodiscarded. The remaining FLK1+ and CD34+ cells are therefore CD43− andCD235A− cells, the desired HEs.

Other methods of generating HEs from ESC and iPSC are known in the art.For example, as described in U.S. Pat. No. 9,382,531, the contents ofwhich is incorporated herein by reference in its entirety.

Endothelial-to-Hematopoietic Transition (EHT)

The endothelial to hematopoietic transition (EHT) is a key developmentalevent leading to the formation of blood stem and progenitor cells duringembryogenesis. In embryogenesis, the development of hematopoietic cellsin the embryo proceeds sequentially from mesoderm through thehemangioblast to the hemogenic endothelium (HE) and hematopoieticprogenitors cells. The HE then undergoes the EHT by becomingpre-hematopoietic stem and progenitor cells (Pre-HSPC). Eventually afterlosing all their endothelial characteristics they become HSPC.

In vitro culture, it is possible to induce EHT in HE with certainfactors and cytokines. Combination of all these factors is required forefficient achievement of EHT.

In one embodiment of any one aspect described, the EHT occurs byculturing or contacting the isolated HE in culture with thrombopoietin(TPO), interleukin (IL)-3, stem cell factor (SCF), IL-6, IL-11,insulin-like growth factor (IGF)-1, erythropoietin (EPO), vascularendothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),bone morphogenetic protein (BMP)4, Fms related tyrosine kinase 3 ligand(Flt-3L), sonic hedgehog (SHH), angiotensin II, and chemical AGTR1(angiotensin II receptor type I) blocker losartan potassium.

In one embodiment of any one aspect described, the isolated HEs areincubated in the EHT media for a period of time.

In one embodiment of any one aspect described, the incubation of theisolated HE in the EHT media is for about 3-10 days.

In one embodiment of any one aspect described, the incubation of theisolated HE in the EHT media is for about 3-9 days, 3-8 days, 3-7 days,3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days,4-6 days, 4-5 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-8 days,5-7 days, 5- 6 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-10 days,7-9 days, 7-8 days, 8-10 days, 8-9 days, or 9-10 days.

In one embodiment of any one aspect described, incubation of theisolated HE in the EHT media is for about 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, or 10 days.

To determine that EHT has occurred in the HE, morpholigical detection ofround cells under microscopy was performed. In addition, FACS analysisof surface markers CD34+CD45+ is performed.

In one embodiment of any one aspect described, the isolated HE that haveundergone EHT exhibit round cell morphology.

Transcription Factors that Induced Differentiation of HE toMulti-Lineage HSCs and HSPCs

To specifying HSCs and HSPCs from the HE having undergone EHT, theinventors use a strategy that is defined as “respecification”.Respecification combines directed differentiation withtranscription-based reprogramming to re-establish HSC fate. Themolecular differences between primary human HSCs and progenitors havebeen well characterized by gene expression profiling, providing arational approach to introduce stem cell genes back into progenitors.The inventors were able to obtain transplantable HSC by restoring theHSC transcription factor network in the HE derived from hPSCs.

The inventors tailored transcription factor combinations for the HE. Theminimally required five transcription factors: ERG, HOXA9, HOXA5, LCORand RUNX1. Additionally, HOXA10 and SPI1 transcription factors can beused to induce differentiation of the HE to multilineage hematopoieticprogenitors.

Generation of iPSCs by somatic cell reprogramming involves globalepigenetic remodeling, and chromatin-modifying enzymes have beencharacterized as barriers or facilitators of reprogramming. Within thehematopoietic system, there are many epigenetic changes that mediateblood development during ontogeny and differentiation from HSCs tomature progeny. The progression from HSCs to differentiated progenyinvolves coordinated control of gene expression programs leading to theactivation or repression of lineage-specific genes. The events that leadto the formation of all mature hematopoietic cells involve regulation ofboth gene expression and DNA recombination, mainly through the controlof chromatin accessibility. HSC state is controlled by a large number oftranscription factors and epigenetic modifiers. The inventors usedscreening strategies find additional factors that regulate of the HSCfate.

Accordingly, in one embodiment of any method, cells, or compositiondescribed herein, the multilineage hematopoietic progenitor cells aregenerated by introducing in vitro an exogenous gene coding copy each ofthe following transcription factors: ERG, HOXA9, HOXA5, LCOR and RUNX1,into the HE, the HE having undergone EHT and exhibit round cellmorphology. In one embodiment, a vector is used as the transport vehicleto introduce any of the herein described exogenous gene coding copiesinto the HE. For example, by transfecting the HE with a vector or more,wherein the vector(s) collectively carry an exogenous gene coding copyof each of the following transcription factors, ERG, HOXA9, HOXA5, LCORand RUNX1, for the in vivo expression of the transcription factor in thetransfected cells. For example, by contacting the HE with a vector ormore, wherein the vector(s) collectively carry an exogenous gene codingcopy of each of the following transcription factors, ERG, HOXA9, HOXA5,LCOR and RUNX1, for the in vivo expression of the transcription factorin the contacted cells. For example, by contacting the isolated HE witha nucleic acid or more, wherein the nucleic acid (s) collectively carryan exogenous gene coding copy of each of the following transcriptionfactors, ERG, HOXA9, HOXA5, LCOR and RUNX1, for the in vivo expressionof the transcription factor in the contacted cells.

In one embodiment of any method, cells, or composition described herein,the multilineage hematopoietic progenitor cells are generated bycontacting a population of HE with a vector or more, wherein thevector(s) collectively carrying an exogenous gene coding copy of each ofthe following transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1,for the in vivo expression of the factors in the contacted cells, andwherein the transfected transcription factors are expressed in vivo inthe contacted cells. The contacting is in vitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the multilineage hematopoietic progenitor cells are generated bycontacting the HE with a nucleic acid or more, wherein the nucleic acid(s) collectively comprises an exogenous gene coding copy of each of thefollowing transcription factors, ERG, HOXA9, HOXA5, LCOR and RUNX1, forthe in vivo expression of the transcription factor in the contactedcells. The contacting is in vitro or ex vivo.

In one embodiment of any method, cells, or composition described herein,the contacting of the HE with any vector(s), nucleic acid(s) orcompositions comprising the vector(s) or nucleic acid(s) describedherein occurs in vitro or ex vivo.

In one embodiment of any methods, cells, or composition describedherein, the contacting or introduction is repeated at least once. In oneembodiment of any methods, cells, or composition described herein, thecontacting or introduction is repeated twice or more times.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factor, HOXA10 or SPI1 or both HOXA10and SPI1, wherein the transfected transcription factor(s) is/areexpressed in vivo in the transfected cells. The transfecting is in vitroor ex vivo.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factors, HOXA10, wherein thetransfected transcription factor is expressed in vivo in the transfectedcells.

In one embodiment of any method, cells, or composition described herein,the method further comprising transfecting the HE with an exogenous genecoding copy of the transcription factors, SPI1, wherein the transfectedtranscription factor is expressed in vivo in the transfected cells.

In one embodiment of any one aspect described, the disclosedtranscription factors are expressed in the transfected cells.

In another embodiment of any one aspect described, the disclosedtranscription factors are expressed in the engineered cells.

In one embodiment of any one aspect described, the expression of thedisclosed transcription factors in the transfected or engineered cellsproduces a population of multi-lineage HSCs and HSPCs.

Transcription Factors

Runt Related Transcription Factor 1 (RUNX1) is the alpha subunit 2 of aheterodimeric transcription factor that binds to the core element ofmany enhancers and promoters. The protein encoded by this gene RUNX1represents the alpha subunit of the heterodimeric transcription factorand is thought to be involved in the development of normalhematopoiesis. Chromosomal translocations involving this gene arewell-documented and have been associated with several types of leukemia.Three transcript variants encoding different isoforms have been foundfor this gene. RUNX1 is essential for hematopoietic commitment of HE andcan convert endothelial cells to hematopoietic progenitor cells. TheREFSEQ mRNAs for RUNX1 are NM_001001890.2; NM_001122607.1; NM_001754.4;XM_005261068.3; ani XM_005261069.4.

Ligand Dependent Nuclear Receptor Corepressor (LCOR) is atranscriptional corepressor widely expressed in fetal and adult tissuesthat is recruited to agonist-bound nuclear receptors through a singleLxxLL motif, also referred to as a nuclear receptor (NR) box. LCOR is acomponent of histone deacetylation complex, is mutated in B-celllymphoma, suggesting a role in B-lymphopoiesis, but this factor has notpreviously been implicated in HSC functions and its role remains to bedefined. LCOR may act as transcription activator that binds DNA elementswith the sequence 5-CCCTATCGATCGATCTCTACCT-3 (By similarity). It is arepressor of ligand-dependent transcription activation by target nuclearreceptors, repressing of ligand-dependent transcription activation byESR1, ESR2, NR3C1, PGR, RARA, RARB, RARG, RXRA and VDR. The REFSEQ mRNAsfor LCOR are NM_015652.3; NM_001170765.1; NM_001170766.1;NM_001346516.1; and NM_032440.3.

ERG (ETS-related gene) is an oncogene meaning that it encodes a proteinthat typically is mutated in cancer. ERG is a member of the ETS(erythroblast transformation—specific) family of transcription factors.The ERG gene encodes for a protein, also called ERG that functions as atranscriptional regulator. Genes in the ETS family regulate embryonicdevelopment, cell proliferation, differentiation, angiogenesis,inflammation, and apoptosis. The external idenifications for ERG geneare as follows: HGNC: 3446; Entrez Gene: 2078; Ensembl: ENSG00000157554;OMIM: 165080; UniProtKB: P11308; EMBL: AY204741 mRNA and thecorresponding mRNA translation: AAP41719.1; and GENBANK: AY204742 mRNAand the corresponding mRNA translation: AAP41720.1.

Spi-1 Proto-Oncogene (SPI1, also known as PU.1) is required forhematopoietic progenitor cell emergence and regulates myeloidspecification. The oncogene is an ETS-domain transcription factor thatactivates gene expression during myeloid and B-lymphoid celldevelopment. The nuclear protein binds to a purine-rich sequence knownas the PU-box found near the promoters of target genes, and regulatestheir expression in coordination with other transcription factors andcofactors. The protein can also regulate alternative splicing of targetgenes. Multiple transcript variants encoding different isoforms havebeen found for this gene. The external idenifications for SPI1 gene isas follows: HGNC: 11241; Entrez Gene: 6688; Ensembl: ENSG00000066336;OMIM: 165170; and UniProtKB: P17947. The REFSEQ mRNAs for SPI1 areNM_001080547.1; NM_003120.2; XM_011520307.1 and XM_017018173.1.

Homeobox protein Hox-A9 is a protein that in humans is encoded by theHOXA9 gene. In vertebrates, the genes encoding the homeobox genes classof transcription factors are found in clusters named A, B, C, and D onfour separate chromosomes. Expression of these proteins is spatially andtemporally regulated during embryonic development. Hox-A9 is part of theA cluster on chromosome 7 and encodes a DNA-binding transcription factorwhich may regulate gene expression, morphogenesis, and differentiation.The external idenifications for HOXA9 gene are as follows: HGNC: 5109;Entrez Gene: 3205; Ensembl: ENSG00000078399; OMIM: 142956; UniProtKB:P31269; EMBL: BT006990 mRNA and the corresponding mRNA translation:AAP35636.1; and GENBANK:AC004080 Genomic DNA. The REFSEQ mRNAs for HOXA9is NM 152739.3

HOX family members have been reproducibly implicated in hematopoiesisacross species. HOXA9 is the key homeotic gene that defines HSCidentity, interacting with ERG to support HSC renewal duringembryogenesis and stress hematopoiesis, suggesting a basis for thefunctional cooperation of HOXA9 and ERG in our system. HOXA5 is atranscriptional target of Notch signaling in T-cell progenitors alongwith HOXA9 and HOXA10, consistent with a role in T-lymphopoiesis. Thesefactors share binding sites in the genome and cooperate to recruitchromatin modulators (e.g. RUNX1 and HOXA families) to induce andmaintain HSPCs. The external idenifications for HOXA5 gene are asfollows: HGNC: 5106; Entrez Gene: 3202; Ensembl: ENSG00000106004; OMIM:142952; and UniProtKB: P20719. The REFSEQ mRNAs for HOXA5 isNM_019102.3. The external idenifications for HOXA5 gene are as follows:HGNC: 5100; Entrez Gene: 3206; Ensembl: ENSG00000253293; OMIM: 142957,and UniProtKB: P31260. The REFSEQ mRNAs for HOXA10 are NM_018951.3 andNM_153715.3.

HOX- and ETS-family transcription factors HOXA9 and ERG are inducers ofself-renewal and multilineage potential in hematopoietic progenitorsdifferentiated from hPSCs. RORA is a nuclear receptor that plays a rolein maintaining quiescence of hematopoietic progenitors. The addition ofSOX4 and MYB modulates this network to enable myeloid and erythroidengraftment in vivo.

OCT4, SOX2, KLF4 and c-MYC are the original four transcription factorsidentified to reprogram mouse fibroblasts into iPSCs. These same fourfactors were also sufficient to generate human iPSCs. OCT3/4 and SOX2function as core transcription factors of the pluripotency network byregulating the expression of pluripotency-associated genes. Krüppel-likefactor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouseES cells and regulates self-renewal. Human iPSCs can also be generatedusing four alternative factors; OCT4 and SOX2 are required but KLF4 andc-MYC could be replaced with NANOG, a homeobox protein important for themaintenance of pluripotency in both ES cells and early embryos, andLIN28, an RNA binding protein.

Transcription factor SOX-4 (SOX4). This intronless gene encodes a memberof the SOX (SRY-related HMG-box) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of the cell fate. The encoded protein act as atranscriptional regulator after forming a protein complex with otherproteins, such as syndecan binding protein (syntenin). The protein mayfunction in the apoptosis pathway leading to cell death as well as totumorigenesis and may mediate downstream effects of parathyroid hormone(PTH) and PTH-related protein (PTHrP) in bone development. The externalidenifications for Homo sapiens (Human) SOX4 gene are as follows: HGNC:11200; Entrez Gene: 6659; Ensembl: ENSG00000124766; OMIM: 184430;UniProtKB: Q06945; EMBL: BC072668 mRNA mRNA and the corresponding mRNAtranslation: AAH72668.1; GENBANK: X65661 mRNA and the corresponding mRNAtranslation: CAA46612.1.

The cDNA encoding the described and desired transcription factors can becloned by methods known in the art into expression vectors for in vivoexpression in the cells. The expression vectors can be constitutive orinducible vectors. The protein and DNA information for transcriptionfactors can be found in the publically available databases such as theGenBank™ database on the National Institute of Health, the UniProt atthe Protein knowledgebase, and GeneCard database at the WeizmannInstitute for Science. The cDNA clones or plasmids carrying the cDNA canbe purchased at BioCat GmbH, and the lentivirus carrying the cDNAs forexpression can also be purchased at Applied Biological Materials (ABM)Inc.

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor into the target cellsselected from the disclosed HE.

In one embodiment of any method, cells, or composition described herein,a vector is used as a transport vehicle to introduce any of the hereindescribed nucleic acid comprising the described exogenous gene codingcopies of transcription factors or reprogramming factors or nucleic acidinhibitor into the target cells selected from the disclosed HE.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopies of each of the transcription factors or reprogramming factors ornucleic acid inhibitor described. The exogenous gene coding copy is forthe expression of transcription factors or reprogramming factors insidethe cells. In one embodiment, each vector consists essentially of atranscription factors or reprogramming factor described herein. In oneembodiment, each vector consists essentially of two or more of thedescribed transcription factors or reprogramming factors.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises nucleic acids comprisingthe described exogenous gene coding copies of transcription factors orreprogramming factors or nucleic acid inhibitor. The nucleic acid is forthe expression of the transcription factors or reprogramming factorsinside the cells.

In one aspect, the present specification provides a vector or more,wherein the vector(s) collectively comprises an exogenous gene codingcopy of each of the following transcription factors, ERG, HOXA9, HOXA5,LCOR and RUNX1 described herein. In another aspect, the vector(s)collectively further comprise an exogenous gene coding copy of HOXA10and SPI1.

In another aspect, the disclosed herein also provides a host cellcomprising a vector or more described herein or nucleic acid(s) of thetranscription factors or reprogramming factors or both described herein.

In another aspect, the disclose also provides a host cell comprising avector or more described herein or nucleic acid(s) of the transcriptionfactors, ERG, HOXA9, HOXA5, LCOR and RUNX1 described herein.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of the transcription factors HOXA10and SPI1.

In another aspect, the host cell further comprises a vector or moredescribed herein or nucleic acid(s) of reprogramming factors or bothdescribed herein, OCT4, SOX2, and KLF4, and optionally with c-MYC ornanog and LIN28. For example, the one or more vectors collectively carrythe nucleic acids of the reprogramming factors OCT4, SOX2, NANOG, andLIN 28, or collectively carry the nucleic acids of the reprogrammingfactors OCT4, SOX2, and KLF4, or OCT4, SOX2, KLF4, and c-MYC.

In one embodiment of any method, cells, or composition described herein,the vector further comprises a spleen focus-forming virus promoter, atetracycline-inducible promoter, a Doxycycline (Dox)-inducible, or aβ-globin locus control region and a β-globin promoter. In oneembodiment, the promoter provide for targeted expression of the nucleicacid molecule therein. Other examples of promoters include but are notlimited to the CMV promoter and EF1α promoters for the varioustransgenes.

In one embodiment of any method, cells, or composition described herein,the vector is a virus.

In one embodiment of any method, cells, or composition described herein,the virus is a lentivirus, an adenovirus, an adeno-associated virus, apox virus, an alphavirus, or a herpes virus. In one embodiment of anymethod, cells, or composition described herein, the virus is an avianviral vector.

In one embodiment of any method, cells, or composition described herein,the in vivo expression of the described transcription factors areregulatable. That is, the promoters used in the vectors for geneexpression are inducible.

In one aspect of any method, cells, or composition described herein, thelentivirus is selected from the group consisting of: humanimmunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equineinfectious anemia virus (EIAV), feline immunodeficiency virus (FIV),bovine immune deficiency virus (BIV), and simian immunodeficiency virus(SIV).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional nucleic acid segments canbe ligated. Another type of vector is a viral vector, wherein additionalnucleic acid segments can be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”,or more simply “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the methods and compositions described herein can include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, lentiviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

Within an expression vector, “operably linked” is intended to mean thatthe nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in atarget cell when the vector is introduced into the target cell). Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).Furthermore, the DNA-targeting endonuclease can be delivered by way of avector comprising a regulatory sequence to direct synthesis of theDNA-targeting endonuclease at specific intervals, or over a specifictime period. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the target cell, the level of expression desired, and the like.

Suitable viral vectors include, but are not limited to, vectors based onRNA viruses, such as retrovirus-derived vectors (for example, Moloneymurine leukemia virus (MLV)-derived vectors), and more complexretrovirus-derived vectors (such as Lentivirus-derived vectors); andvectors based on DNA viruses, such as adenovirus-based vectors andadeno-associated virus (AAV)-based vectors. In some embodiments, thepolynucleotide delivery system comprises a retroviral vector, morepreferably a lentiviral vector. Non-limiting examples of viral vectorinclude lentivirus vectors derived from human immunodeficiency virus 1(HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectiousanemia virus, simian immunodeficiency virus (SIV) and maedi/visna virus.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,engrafts in vivo in the recipient subject and produces blood cells invivo.

In one embodiment of any one aspect described, the population ofmulti-lineage HSCs and HSPCs, produced by the expression of thedisclosed transcription factors in the transfected or engineered cells,reconstitutes the hematopoietic system in vivo in the recipient subject.

In one embodiment of any one aspect described, the engineered cellcomprises an exogenous copy of each of the following transcriptionfactors ERG, HOXA9, HOXA5, LCOR and RUNX1.

In one embodiment of any one aspect described, the engineered cellfurther comprises an exogenous copy of each of the followingreprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC.

In one embodiment of any one aspect described, the engineered cellfurther comprises an exogenous copy of each of the followingreprogramming factors OCT 4, SOX2, NANOG, and LIN28.

In one embodiment of any one aspect described, the composition ofengineered cells further comprises a pharmaceutically acceptablecarrier.

In one embodiment of any one aspect described, the subject is patientswho has undergone chemotherapy or irradiation or both, and manifestdeficiencies in immune or blood function or lymphocyte reconstitution orboth deficiencies in immune function and lymphocyte reconstitution.

In one embodiment of any one aspect described, the subject prior toimplantation, the immune cells are treated ex vivo with prostaglandin E2and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequentengraftment in a recipient subject.

In one embodiment of any one aspect described, the disclosed engineeredcells are autologous to the recipient subject or at least HLA typematched with the recipient subject.

It is also envisioned that the methods described herein can be used asprophylaxis

Uses of Engineered Immune Cells Derived from Pluripotent Stem Cells

The engineered cells described herein are useful in the laboratory forbiological studies. For examples, these cells can be derived from anindividual having a genetic disease or defect, and used in thelaboratory to study the biological aspects of the diseases or defect,and to screen and test for potential remedy for that diseases or defect.Contemplated but not limited to are congenital hematologic disorderssuch as Diamond-Blackfan-Anemia, Shwatchman-Diamond-Anemia, and immunedeficiency such as Omenn syndrome.

Alternatively, the engineered cells described herein are useful incellular replacement therapy in subjects having the need. For example,patients who have undergone chemotherapy. For example, patients who haveundergone chemotherapy or irradiation or both, and manifest deficienciesin immune function and/or lymphocyte reconstitution. For example,cellular replacement therapy can be performed in subjects withcongenital hematologic disorders described herein.

In various embodiments, the engineered cells described herein areadministered (ie. implanted or transplanted) to a subject in need ofcellular replacement therapy. The implanted engineered cell engrafts andreconstitutes the hematopoietic system in the recipient subject.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof described cells, e.g. HSC, HSPC, hematopoietic progenitor cells, intoa subject, by a method or route which results in at least partiallocalization of the introduced cells at a desired site, such as a siteof injury or repair, such that a desired effect(s) is produced. Thecells e.g. HSC, HSPC, hematopoietic progenitor cells, or theirdifferentiated progeny can be administered by any appropriate routewhich results in delivery to a desired location in the subject where atleast a portion of the implanted cells or components of the cells remainviable.

In various embodiments, the engineered cells described herein areoptionally expanded ex vivo prior to administration to a subject. Inother embodiments, the engineered cells are optionally cryopreserved fora period, then thawed prior to administration to a subject.

The engineered cells used for cellular replacement therapy can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic) in relation to the recipient of thecells. “Autologous,” as used herein, refers to cells from the samesubject. “Allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison. “Syngeneic,”as used herein, refers to cells of a different subject that aregenetically identical to the cell in comparison. “Xenogeneic,” as usedherein, refers to cells of a different species to the cell incomparison. In other embodiments, the engineered cells of theembodiments of the present disclosure are allogeneic.

In various embodiments, the engineered cell described herein that is tobe implanted into a subject in need thereof is autologous or allogeneicto the subject.

In various embodiments, the engineered cell described herein can bederived from one or more donors, or can be obtained from an autologoussource. In some embodiments of the aspects described herein, theengineered cells are expanded in culture prior to administration to asubject in need thereof.

In various embodiments, the engineered cell described herein can bederived from one or more donors, or can be obtained from an autologoussource.

In various embodiments, prior to implantation, the recipient subject istreated, or was previously been treated with chemotherapy and/orradiation.

In one embodiment, the chemotherapy and/or radiation are to reduceendogenous stem cells in the recipient subject to facilitate engraftmentof the implanted cells.

In one embodiment, the chemotherapy and/or radiation have reduced theability of the recipient subject to synthesize sufficient hematopoieticcells to sustain life, e.g., inability to make blood cells, plateletcells, etc.

In various embodiments, prior to implantation, the engineered immunecells or the inhibited, reverse-lineage multilineage hematopoieticprogenitor cells are treated ex vivo with prostaglandin E2 and/orantioxidant N-acetyl-L-cysteine (NAC) to promote subsequent engraftmentin a recipient subject.

In various embodiments, the recipient subject is a human.

In various embodiments, the subject is diagnosed with HIV, ahematological disease, undergoing a cancer treatment, or an autoimmunedisease. For example, the subject has aplastic anemia, X-linkedlymphopenic immune deficiency, Wiskott-Aldrich syndrome, Fanconi anemia,cancer, or acute lymphoblastic leukemia (ALL).

In one aspect of any method, cells and composition described herein, asubject is selected to donate a somatic cell which would be used toproduce iPSCs and an engineered immune cell described herein. In oneembodiment, the selected subject has a genetic disease or defect.

In various embodiments, the donor subject is a human.

In various embodiments, the donor or the recipient subject is an animal,human or non-human, and rodent or non-rodent. For example, the subjectcan be any mammal, e.g., a human, other primate, pig, rodent such asmouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheepor goat, or a non-mammal such as a bird.

In various embodiments, the donor or the recipient subject is diagnosedwith HIV, a hematological disease or cancer.

In one aspect of any method, cells and composition described herein, abiological sample or a population of embryonic stem cells, somatic stemcells, progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells is obtained from the donor subject.

In various embodiments, biological sample or a population of embryonicstem cells, somatic stem cells, progenitor cells, bone marrow cells,hematopoietic stem cells, or hematopoietic progenitor cells describedherein can be derived from one or more donors, or can be obtained froman autologous source.

In one embodiment, the embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells,hematopoietic progenitor cells are isolated from the donor subject,transfected, cultured (optional), and transplanted back into the samesubject, i. e. an autologous cell transplant. Here, the donor and therecipient subject is the same individual. In another embodiment, theembryonic stem cells, somatic stem cells, progenitor cells, bone marrowcells, hematopoietic stem cells, or hematopoietic progenitor cells areisolated from a donor who is an HLA-type match with a subject(recipient). Donor-recipient antigen type-matching is well known in theart. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. Theserepresent the minimum number of cell surface antigen matching requiredfor transplantation. That is the transfected cells are transplanted intoa different subject, i.e., allogeneic to the recipient host subject. Thedonor's or subject's embryonic stem cells, somatic stem cells,progenitor cells, bone marrow cells, hematopoietic stem cells, orhematopoietic progenitor cells can be transfected with a vector ornucleic acid comprising the nucleic acid molecule(s) described herein,the transfected cells are cultured, inhibited, and differentiated asdisclosed, optionally expanded, and then transplanted into the recipientsubject. In one embodiment, the transplanted engineered immune cellsengrafts in the recipient subject. In one embodiment, the transplantedengineered immune cells reconstitute the immune system in the recipientsubject. The transfected cells can also be cryopreserved aftertransfected and stored, or cryopreserved after cell expansion andstored.

The engineered cells having multilineage hematopoietic progenitor cellsdescribed herein may be administered as part of a bone marrow or cordblood transplant in an individual that has or has not undergone bonemarrow ablative therapy. In one embodiment, genetically modified cellscontemplated herein are administered in a bone marrow transplant to anindividual that has undergone chemoablative or radioablative bone marrowtherapy.

In one embodiment, a dose of cells is delivered to a subjectintravenously. In one embodiment, the cells are intravenouslyadministered to a subject.

In particular embodiments, patients receive a dose of the cells, e.g.,engineered cells of this disclosure, of about 1×10⁵ cells/kg, about5×10⁵ cells/kg, about 1×10⁶ cells/kg, about 2×10⁶ cells/kg, about 3×10⁶cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about 6×10⁶cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, about 1×10⁷ cells/kg, about 5×10⁷ cells/kg, about 1×10⁸cells/kg, or more in one single intravenous dose.

In certain embodiments, patients receive a dose of the cells, e.g.,engineered cells, of at least 1×10⁵ cells/kg, at least 5×10⁵ cells/kg,at least 1×10⁶ cells/kg, at least 2×10⁶ cells/kg, at least 3×10⁶cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg, at least6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶ cells/kg, atleast 9×10⁶ cells/kg, at least 1×10⁷ cells/kg, at least 5×10⁷ cells/kg,at least 1×10⁸ cells/kg, or more in one single intravenous dose.

In an additional embodiment, patients receive a dose of the cells, e.g.,engineered cells of this disclosure, of about 1×10⁵ cells/kg to about1×10⁸ cells/kg, about 1×10⁶ cells/kg to about 1×10⁸ cells/kg, about1×10⁶ cells/kg to about 9×10⁶ cells/kg, about 2×10⁶ cells/kg to about8×10⁶ cells/kg, about 2×10⁶ cells/kg to about 8×10⁶ cells/kg, about2×10⁶ cells/kg to about 5×10⁶ cells/kg, about 3×10⁶ cells/kg to about5×10⁶ cells/kg, about 3×10⁶ cells/kg to about 4×10⁸ cells/kg, or anyintervening dose of cells/kg.

In general, the engineered cells described herein are administered as asuspension with a pharmaceutically acceptable carrier. For example, astherapeutic compositions. Therapeutic compositions contain aphysiologically tolerable carrier together with the cell composition andoptionally at least one additional bioactive agent as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not substantially immunogenicwhen administered to a mammal or human patient for therapeutic purposes,unless so desired. One of skill in the art will recognize that apharmaceutically acceptable carrier to be used in a cell compositionwill not include buffers, compounds, cryopreservation agents,preservatives, or other agents in amounts that substantially interferewith the viability of the cells to be delivered to the subject. Aformulation comprising cells can include e.g., osmotic buffers thatpermit cell membrane integrity to be maintained, and optionally,nutrients to maintain cell viability or enhance engraftment uponadministration. Such formulations and suspensions are known to those ofskill in the art and/or can be adapted for use with the cells asdescribed herein using routine experimentation.

In some embodiments, the compositions of engineered cells describedfurther comprise a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutically acceptable carrier does not includetissue or cell culture media.

In various embodiments, a second or subsequent dose of cells isadministered to the recipient subject. For example, second andsubsequent administrations can be given between about one day to about30 weeks after the previous administration. Two, three, four or moretotal administrations can be delivered to the individual, as needed.

A cell composition can be administered by any appropriate route whichresults in effective cellular replacement treatment in the subject, i.e.administration results in delivery to a desired location in the subjectwhere at least a portion of the composition delivered, i.e. at least1×10⁴ cells are delivered to the desired site for a period of time.Modes of administration include injection, infusion, or instillation,“Injection” includes, without limitation, intravenous, intra-arterial,intraventricular, intracardiac injection and infusion. For the deliveryof cells, administration by injection or infusion is generallypreferred.

Efficacy testing can be performed during the course of treatment usingthe methods described herein. Measurements of the degree of severity ofa number of symptoms associated with a particular ailment are notedprior to the start of a treatment and then at later specific time periodafter the start of the treatment.

The embodiments of the present disclosure can be defined in any of thefollowing numbered paragraphs:

-   -   [1]. A method for making hematopoietic stem cells (HSCs) and        hematopoietic stem and progenitor cells (HSPCs) comprising in        vitro transfecting hemogenic endothelia cells (HE) with an        exogenous gene coding copy of each of the following        transcription factors ERG, HOXA9, HOXA5, LCOR and RUNX1, wherein        the transcription factors are expressed in the transfected cells        to produce a population of multilineage HSCs and HSPCs that        engrafts in recipient host after implantation.    -   [2]. A method of making hematopoietic stem cells (HSCs) and        hematopoietic stem and progenitor cells (HSPCs) comprising: (a)        generating embryonic bodies (EB) from pluripotent stem        cells; (b) isolating hemogenic endothelia cells (HE) from the        resultant population of EB; (c) inducing        endothelial-to-hematopoietic transition (EHT) in culture in the        isolated HE to obtain hematopoietic stem cells, and (d) in vitro        transfecting the induced HE with an exogenous gene coding copy        of each of the following transcription factors ERG, HOXA9,        HOXA5, LCOR and RUNX1.    -   [3]. The method of paragraph 1 or 2, wherein the method is an in        vitro method.    -   [4]. The method of any one of the preceding paragraphs, wherein        the EB are generated or induced from pluripotent stem cells        (PSC) by culturing or exposing the PSC to mophogens for about 8        days.    -   [5]. The method of paragraph 4, wherein the mophogens selected        from the group consisting of Holo-Transferrin, mono-thioglycerol        (MTG), ascorbic acid, bone morphogenetic protein (BMP)-4, basic        fibroblast growth factor (bFGF), SB431542, CHIR99021, vascular        endothelial growth factor (VEGF), interleukin (IL)-6,        insulin-like growth factor (IGF)-1, interleukin (IL)-11, stem        cell factor (SCF), erythropoietin (EPO), thrombopoietin (TPO),        interleukin (IL)-3, and Fms related tyrosine kinease 3 ligand        (Flt-3L).    -   [6]. The method of any one of the preceding paragraphs, wherein        the EBs are less than 800 microns in size and are selected.    -   [7]. The method of any one of the preceding paragraphs, wherein        the EB cells within the EBs are compactly adhered to each other        and requires trypsin digestion in order to dissociate the cells        to individual cells.    -   [8]. The method of paragraph 5 or 7, wherein the EB cells of the        selected EBs are dissociated prior to the isolation of HE.    -   [9]. The method of any one of the preceding paragraphs, wherein        the population of PSC is induced pluripotent stem cells (iPS        cells) or embryonic stem cells (ESC).    -   [10]. The method of paragraph 9, wherein the induced pluripotent        stem cells are produced by introducing only reprogramming        factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28        into mature cells.    -   [11]. The method of paragraph 10, wherein the mature cells are        selected from the group consisting of B lymphocytes (B-cells), T        lymphocytes, (T-cells), fibroblasts, and keratinocytes.    -   [12]. The method of paragraph 8, 9 or 10, wherein the induced        pluripotent stem cells are produced by introducing the        reprogramming factors two or more times into the mature cells.    -   [13]. The method of any one of the preceding paragraphs, wherein        the HE are definitive HE.    -   [14]. The method of any one of the preceding paragraphs, wherein        the HE are isolated immediately from selected and dissociated        EB.    -   [15]. The method of any one of the preceding paragraphs, wherein        the HE are FLK1+, CD34+, CD43−, and CD235A−.    -   [16]. The method of any one of the preceding paragraphs, wherein        the hematopoietic cells are CD34+ and CD45+.    -   [17]. The method of any one of the preceding paragraphs, wherein        the endothelial-to-hematopoietic transition occurs by culturing        the isolated HE in thrombopoietin (TPO), interleukin (IL)-3,        stem cell factor (SCF), IL-6, IL-11, insulin-like growth factor        (IGF)-1, erythropoietin (EPO), vascular endothelial growth        factor (VEGF), basic fibroblast growth factor (bFGF), bone        morphogenetic protein (BMP)4, Fms related tyrosine kinase 3        ligand (Flt-3L), sonic hedgehog (SHH), angiotensin II, chemical        AGTR1 (angiotensin II receptor type I) blocker losartan        potassium.    -   [18]. The method of any one of the preceding paragraphs, wherein        the multilineage HSCs are CD34+CD38−CD45+.    -   [19]. The method of any one of the preceding paragraphs, wherein        the multilineage HSPCs are CD34+CD45+.    -   [20]. An engineered cell derived from a population of HE and        produced by a method of any one of paragraphs 1-18.    -   [21]. The engineered cell of paragraph 19, wherein the        engineered cell comprises an exogenous copy of each of the        following transcription factors ERG, HOXA9, HOXA5, LCOR and        RUNX1.    -   [22]. The engineered cell of paragraph 19 or 20, wherein the        engineered cell further comprises an exogenous copy of each of        the following reprogramming factors OCT4, SOX2, KLF4 and        optionally c-MYC.    -   [23]. A composition comprising a population of engineered cells        of any one of paragraphs 19-21.    -   [24]. The composition of paragraph 22, further comprising a        pharmaceutically acceptable carrier.    -   [25]. A pharmaceutical composition comprising a population of        engineered cells of any one of paragraphs 19-21 and a        pharmaceutically acceptable carrier.    -   [26]. A pharmaceutical composition of paragraph 24 for use in        cellular replacement therapy in a subject.    -   [27]. A method of cellular replacement therapy in a subject in        need thereof, the method comprising administering a population        of engineered cells of any one of the preceding paragraphs        19-21, or a composition of claim 22-23, or a pharmaceutical        composition of any one of the preceding paragraphs 24-25 to a        recipient subject.    -   [28]. The method of cellular replacement therapy of paragraph        27, wherein the subject is a patient who has undergone        chemotherapy or irradiation or both, and manifest deficiencies        in immune function or lymphocyte reconstitution or both        deficiencies in immune function and lymphocyte reconstitution.    -   [29]. The method of cellular replacement therapy of paragraph 27        or 28, wherein the subject prior to implantation, the immune        cells are treated ex vivo with prostaglandin E2 and/or        antioxidant N-acetyl-L-cysteine (NAC) to promote subsequent        engraftment in a recipient subject.    -   [30]. The method of cellular replacement therapy of paragraph 27        or 28 or 29, wherein the immune cells are autologous to the        recipient subject or at least HLA type matched with the        recipient subject.

The embodiments of the present disclosure are further illustrated by thefollowing example which should not be construed as limiting. Thecontents of all references cited throughout this application, as well asthe figures and table are incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the present disclosure described herein. Such equivalentsare intended to be encompassed by the following claims.

EXAMPLE

Materials and Methods

hPSC Culture

All experiments were performed with H9 hESC (NIHhESC-10-0062), PB34iPS⁵⁹, MSC-IPS1⁶⁰, PB34 iPS⁶⁵, MSC-iPS1⁶⁶, 1045-iPSC, and 1157-iPSCestablished by hES (human embryonic stem cell) core facility at BostonChildren's Hospital. See the Children's Hospital webpage at stemcellperiod childrenshospital period org. Human embryonic stem cells (ESCs)and induced pluripotent stem cells (iPSCs) were maintained on mouseembryonic fibroblasts (GLOBALSTEM) feeders in DMEM/F12+20% KNOCKOUTSERUMReplacement (INVITROGEN), 1 mM L-glutamine, 1 mM NEAA, 0.1 mMbeta-mercaptoethanol, and 10 ng/ml bFGF on 10 cm gelatinized culturedish. Media was changed daily, and cells were passaged 1:4 onto freshfeeders every 7 days using standard clump passaging with collagenase IV.Morphology of pluripotent stem cells (PSCs) was checked under microscopydaily. As a quality control, only dishes with more than 70% of typicalPSC colonies were processed for embryoid bodies (EBs) formation. EBs arethree-dimensional aggregates of pluripotent stem cells. Cell lines weretested for mycoplasma routinely.

EB Differentiation

EB differentiation was performed as previously described¹⁹. Briefly,human pluripotent stem cells (hPSCs) colonies were dissociated with0.05% Trypsin for 5 min in at 37° C., pipetted thoroughly with p1000until single cells or small aggregates, washed twice with PBS with 2%fetal bovine serum (FBS), and re-suspended in StemPro-34-based mediaprior to seeding. StemPro-34 (INVITROGEN, 10639-011) is supplementedwith L-glutamine (2 mM), penicillin/streptomycin (10 ng/mL), ascorbicacid (1 mM), human holo-Transferrin (150 μg/mL, SIGMA T0665), andmonothioglycerol (MTG, 0.4 mM) (referred to as “SupplementedStemPro-34), BMP-4 (10 ng/mL) and Y-27632 (10 uM). The suspended cellswere seeded into non-adherent spheroid formation 10 cm plates(EZSPHERE™, ASAHI GLASS CO; Well Size (μm) Diameter: 400-500, Depth:100-200; No. of well 14,000/dish) at a cell density of 2-5 millioncells/dish. About 24 h after seeding, bFGF (5 ng/mL) and BMP4 (10 ng/mL)were added to the media. At day 2 in culture, the developing EBs werecollected and re-suspended in supplemented StemPro-34 with SB431542 (6μM), CHIR99021 (3 μM), bFGF (5 ng/mL) and BMP4 (10 ng/mL). The formationof EBs were checked under microscopy at day 4 and the decision was madeto continue EB formation based on the size and morphology ofaggregations (quality control; >100 uM, compaction-like tight contact ofcells). At day 4, the media is replaced by supplemented StemPro-34 withVEGF (15 ng/mL) and bFGF (10 ng/mL). At day 6, the media is replaced bysupplemented StemPro-34 with bFGF (10 ng/mL), VEGF (15 ng/mL), IL-6 (10ng/mL), IGF-1 (25 ng/mL), IL-11 (5 ng/mL) and SCF (50 ng/mL). Cultureswere maintained in a 5% CO₂/5% O₂/90% N₂ environment. After 8 days inculture, the EBs are harvested and sorted for definitive HE via surfacemarkers.

HE Sorting

To avoid potential damage with hydrodynamic pressure and contaminationthrough FACS (Elliot et al., 2009), for functional assay, the isolationof HE was carried out by MACS. Freshly dissociated EB cells (at day 8time point) were filtered through a 70 μm filter and stained with CD34microbeads (MILTENYI) for 30 min at 4° C. CD34+ cells were isolated withLS columns (MILTENYI). Around 0.1-0.5×10⁶ cells were obtained per 10 cmdish of EB formation. A sample from each batch was analyzed by FACS tovalidate its purity of HE. The following antibodies or dyes are used inthe FACS: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD),CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD)and DAPI. For accurate isolation of HE for expression profile bymicroarray and qRT-PCR, isolation of HE was carried out by FACS.Dissociated EBs (at day 8 time point) were re-suspended at 1-3×10⁶ per100 ml staining buffer (PBS+2% FBS). Cells were stained with a 1:50dilution of CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD),CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD)and DAPI for 30 min at 4° C. dark. All FACS sorting was performed on aBD FACS Aria II cell sorter using a 100 micrometer nozzle to avoidpotential damage to HE.

Microarray and CellNet Analysis of HE

All the samples used for microarray analysis were FACS sorted. Thefollowing antibodies are used for identifying HE: CD34 PE-Cy7 (8G12;BD), FLK1 AF647 (89106; BD), CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6;Coulter), CD43 PE (1G10; BD). Fetal liver hematopoietic stem cells(HSCs) were purchased from STEMCELL Technologies and stained with thefollowing antibodies for identifying HSC: CD38 PE-Cy5 (LS198-4-3;Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Between 10,000-50,000cells were sorted for each cell type for each replicate (n=2 or 3). TheRNAeasy Microkit (QIAGEN) was used to collect and prepare total RNA formicroarray analysis. The Ovation Picokit (NUGEN) was used forpre-amplification, where required. Gene expression profiling wasperformed on AFFYMETRIX 430 2.0 gene chips per standard protocol.Microarray data were analyzed per standard protocol usingR/Bioconductor. The classification and GRN of HE were analyzed byCellNet (Callan et al., 2014). Briefly, we reasoned that the extent towhich a GRN is established in a sample is reflected in the expression ofthe genes in the GRN such that the GRN genes should fall within a rangeof expression observed in the corresponding C/T in the training data. Weformalized this notion as a GRN status metric, defined in comparison tothe complete training data set. The status of C/T GRN in a query sampleis defined as the weighted mean of the Z scores of the genes in the GRN,where the Z score is defined in reference to the expression distributionof each gene in a C/T. The GRN status score can be weighted by theabsolute expression level of each gene in a C/T so that genes morehighly expressed have more influence on the GRN status (default) and/orby the importance to the Random Forest classifier.

Endothelial-to-Hematopoietic Transition (EHT) Culture

EBs were dissociated on day 8 by digestion with 0.05% Trypsin for 5 minat 37° C., pipetted thoroughly with p1000 pipette to generate a singlecell suspension and washed with PBS+2% FBS. Dissociated EBs wereimmediately processed for the isolation of HE. Unlike HSPCs in previousreport that used frozen batches¹⁷, HE delayed (approximately 2-3 days)recovery of morphology and growth once frozen, thus all the experimentswere done with freshly isolated HE. Thus, cells were re-suspended in 1mL of PBS+2% FBS and incubated with human CD34 MicroBead kit for 1 h(MILTENYL BIOTEC, 130-046-702). After incubation, cells were washed withPBS+2% FBS and isolated by magnetic cell isolation (MACS) using LScolumns (MILTENYL BIOTEC, 130-042-401). Sorted CD34+ cells werere-suspended in supplemented StemPro-34 media, containing Y-27632 (10uM), TPO (30 ng/mL), IL-3 (10 ng/mL), SCF (50 ng/mL), IL-6 (10 ng/mL),IL-11 (5 ng/mL), IGF-1 (25 ng/mL), VEGF (5 ng/mL), bFGF (5 ng/mL), BMP4(10 ng/mL), and Flt-3L (10 ng/mL) as reported²⁰ and seeded at a densityof 25-50×10⁴ cells per well onto thin-layer MATRIGEL-coated 24-wellplates. All recombinant factors are human and most were purchased fromPeprotech.

Sorted HE cells were seeded onto thin-layer MATRIGEL-coated 96-wellplates (flat-bottom) at a density of 5×10⁴/well in supplementedStemPro-34 media, containing TPO (30 ng/ml), IL-3 (30 ng/ml), SCF (100ng/ml), IL-6 (10 ng/ml), IL-11 (5 ng/ml), IGF-1 (25 ng/ml), EPO (2U/ml), VEGF (5 ng/ml), bFGF (5 ng/ml), BMP4 (10 ng/ml), Flt-3L (10ng/ml), SHH (20 ng/ml), angiotensin II (10 μg/l) and the chemical AGTR1(angiotensin II receptor type I) blocker losartan potassium (100 μM,Tocris) as reported²⁰. All recombinant factors are human and most werepurchased from Peprotech. The exception is angiotensin II, which waspurchased from Sigma.

Lentivirus Production

Plasmids for the TF library were obtained as Gateway plasmids (HarvardPlasmid Serive; GeneCopoeia). Open reading frames were cloned intolentiviral vectors using LR Clonase (INVITROGEN). Two vectors were used,pSMAL-GFP (constitutive) and pINDUCER-21⁶¹. Lentiviral particles wereproduced by transfecting 293T-17 cells (ATCC) with the second-generationpackaging plasmids (pMD2.G and psPAX2 from Addgene). Virus was harvested36 and 60 hr after transfection and concentrated by ultracentrifugationat 23,000 rpm for 2 hr 15 min at 4° C. Virus was reconstituted with 50uL of EHT culture media. Constructs were titered by serial dilution on293T cells using GFP as an indicator. Polycistronic vectors were made asfollows. LCOR-P2A-HOXA9-T2A-HOXA5 and RUNX1-P2A-ERG DNA fragments weresynthesized and cloned into TOPO-D cloning vector respectively byGENSCRIPT, then Gateway-recombined with pINDUCER-21.

Lentiviral Gene Transfer

At day 3 of EHT culture, HE cells were beginning to produce potentiallyhematopoietic ‘round’ cells, the occurrence of this phenomenon used asquality control of HE induction and transition to hematopoietic cellsfor each batch of experiment. The infection media was EHT culture mediasupplemented with Polybrene (8 ug/mL, SIGMA). Lentiviral infections werecarried out in a total volume of 150 ul or 250 ul (for a 24-well plate).The multiplicity of infection for the factors was as follows: Library3.0 for each, ERG 5.0, HOXA5 5.0, HOXA9 5.0, HOXA10 5.0, LCOR 5.0, RUNX15.0, and SPI1 5.0. HE was vulnerable to damage during spinoculation,thus infections were carried out static for 12 hours, then 50 uL or 250uL of fresh EHT media was supplemented to dilute Polybrene. Parallelwells were kept cultured for additional 3 days to measure infectionefficiency by percentage of GFP+ DAPI-cells by FACS, 30-70% infectionefficiency.

In Vitro Screening Via CFU

Followed by lentiviral gene transfer, cells were maintained for 5 daysin EHT culture media supplemented with doxycycline (2 ug/mL, SIGMA) toinduce transgene expression in vitro. 5×10⁴ cells were plated into 3 mlof complete methylcellulose (H4434; STEMCELL Technologies). Additionalcytokines added were: 10 ng/ml FLT3, 10 ng/ml IL6, and 50 ng/ml TPO (R&DSystems). The mixture was distributed into two 60 mm dishes andmaintained in a humidified chamber at 37° C. for 14 days. Colonies werescored manually or using the BD Pathway 855 fluorescent imager. At 14days, granurocyte/erythrocyte macrophage/mega-karyocyte (GEMM) colonieswere picked up by P20 pipette. Between 10-20 GEMM colonies were pickedfor each replicates (n=3). The QIAamp DNA Micro kit (QIAGEN) was used tocollect and prepare total genomic DNA for PCR detection of transgenes.Nested PCR reactions were: 1st round with LNCX Fwd primer (5′-AGC TCGTTT AGT GAA CCG TCA GAT C-3′) and EGFP N Rev primer (5′-CGT CGC CGT CCAGCT CGA CCA G-3′). This PCR program consists of 1 cycle of 95° C. for 5min, 36 cycles of 95° C. for 30 sec followed by 60° C. for 30 sec thenfollowed by 72° C. for 5 min, 1 cycle of 72° C. for 10 min, and aterminating cycle of 4° C. for indefinite period; 2nd round with forwardprimer for each gene (listed in Table) and HA Rev primer (5′-TCT GGG ACGTCG TAT GGG TA-3′). This PCR program consists of 1 cycle of 95° C. for 5min, 36 cycles of 95° C. for 30 sec followed by 60° C. for 30 sec thenfollowed by 72° C. for 30 sec, 1 cycle of 72° C. for 10 min, and aterminating cycle of 4° C. for indefinite period.

In Vivo Screening Via Transplantation

12 hours after lentiviral gene transfer, cells were recovered by dispasefor 5 min at 37° C., and washed by PBS three times to ensure nocarry-over of virus. Cells were re-suspended at 0.5-1.0×10⁶ cells per 25uL buffer (PBS+2% FBS from STEMCELL Technologies) and kept on ice untilinjection. 0.5-1.0×10⁶ cells were intrafemorally injected toNOD/LtSz-scidIL2Rgnull (NSG) mice and treated with doxycycline asdescribed in the section on Mouse transplantation. Up to 100 uLperipheral blood (PB) were collected every 2-4 weeks, through 14 weeks.Mice were sacrificed and harvested for bone marrow (BM) and thymus at8-14 weeks. For transgene detection in engrafted cells, each lineagecells were FACS sorted from BM. The following antibodies are used foridentifying cell types, myeloid cells: CD33 APC (P67.6; BD), CD45 PE-Cy5(J33; Coulter). B-cells: CD19 PE (HIB19; BD), CD45 PE-Cy5 (J33;Coulter); T-cells: CD3 PE-Cy7 (SK7; BD), CD45 PE-Cy5 (J33; Coulter).Between 10,000-50,000 cells were isolated with 2 or 3 biologicalreplicates for multiple cell lines (iPSCs and ESCs). The QIAamp DNAMicro kit (QIAGEN) was used to collect and prepare total genomic DNA forPCR detection of transgenes. Nested PCR reaction was done as similar toin vitro screening of above section.

Mouse Transplantation

NOD/LtSz-scidIL2Rgnull (NSG) (Jackson Labs) mice were bred and housed atthe Boston Children's Hospital (BCH) animal care facility. Animalexperiments were performed in accordance to institutional guidelinesapproved by BCH animal care committee. Intra-femoral transplants havebeen previously described. Briefly, 6-10 week old mice were irradiated(250 rads) 12 hrs before transplant. Prior to transplantation, mice weretemporarily sedated with isoflurane. A 27 g needle was used to drill theleft femur (injected femur), and 0.5-1.0×10⁶ cells were transplanted ina 25 μL volume using a 28.5 g insulin needle. Sulfatrim was administeredin drinking water to prevent infections after irradiation. 625 ppmDoxycycline Rodent Diet (Envigo-Teklad Diets) and Doxycycline (1.0mg/ml) was added to the drinking water to maintain transgene expressionin vivo for 2 weeks¹². Secondary transplantation was carried with1,000-2,000 human CD34+ cells (isolated from BM by MACS with CD34microbeads) at 8 weeks. Isolated cells were resuspended at 1,000-2,000cells per 25 uL buffer (PBS+2% FBS from STEMCELL Technologies) and kepton ice until injection. Cells were intrafemorally injected toNOD/LtSz-scidIL2Rgnull (NSG) and treated with Doxycycline via food anddrinking water for 2 weeks.

Flow Cytometry

Cells grown in EHT culture or harvested animal tissues were stained withthe following antibody panels. HE panel: CD34 PE-Cy7 (8G12; BD), FLK1AF647 (89106; BD), CD235a/Glycophorin (GLY)-A FITC (11E4B-7-6; Coulter),CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/Glycophorin(GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter),CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD),TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD) andCD31/PECAM PE (WM59; BD). All stains were performed with <1×106 cellsper 100 μl staining buffer (PBS+2% FBS) with 1:100 dilution of eachantibody, 30 min at 4C in dark. Compensation was performed by automatedcompensation with anti-mouse Igk negative beads (BD) and CB MNC stainedwith individual ab. All acquisition was performed on BD Fortessacytometer. For detection of engraftment, human cord blood-engraftedmouse marrow was used as a control to set gating; Sorting was performedon a BD FACS Aria II cell sorter.

Cytospin of Erythroid and Neutrophils

5,000-10,000 of FACS sorted erythroid (CD235a/Glycophorin (GLY)-A PE-Cy7or FITC (11E4B-7-6; Coulter)), plasmacytoid lymphocytes (CD19 PE (HIB19;BD), IgM BV510 (G20-127; BD), CD38 PE-Cy5 (LS198-4-3; Clontech)) andneutrophils (CD15 APC (HI98; BD), CD31/PECAM PE (WM59; BD) and CD45PE-Cy5 (J33; Coulter)) were cytospun onto slides (500 rpm for 10minutes), air dried, and stained with May-Grunwald and Giemsa stains(both from Sigma), washed with water, air dried, and mounted, followedby examination by light microscopy.

Quantitative PCR

RNA extraction was performed using the RNAeasy Microkit (QIAGEN).Reverse transcription was performed using Superscript III (>5,000 cells)or VILO reagent (<5,000 cells) (Invitrogen). Quantitative PCR wascarried out in triplicate with SYBR Green (Applied Biosystems).Transcript abundance was calculated using the standard curve method.Primers used for globin genes62: huHbB F (5′-CTG AGG AGA AGT CTG CCGTTA-3′), huHbB R (5′-AGC ATC AGG AGT GGA CAG AT-3′), huHbG F (5′-TGG ATGATC TCA AGG GCA C-3′), huHbG R (5′-TCA GTG GTA TCT GGA GGA CA-3′), huHbEF (5′-GCA AGA AGG TGC TGA CTT CC-3′), huHbE R (5′-ACC ATC ACG TTA CCCAGG AG-3′).

ELISA of Terminally Differentiated Cells

FACS isolated neutrophils (CD15+ PECAM+CD45+), T-cells (CD3+CD45+) werecultured in IMDM+10% FBS overnight in 96-well pate (flat-bottom). Thensupernatant was taken and analyzed by MPO- or IFNγ-ELISA-Ready-SET-Go!Kit (eBioscience) according to the manufacturer's protocol. The amountof IFNγ was normalized per 1,000 cells. Human Ig production was measuredfrom 50 uL of serum from NSG mice at 8 weeks (IgM) and 14 weeks postengraftment (IgG). Immunization of mice was done with OVA (F5503, Sigma)with Freund's complete adjuvant (F5881, Sigma), followed by boosterdoses of Freund's incomplete adjuvant (F5506, Sigma) according tomanufacture's instruction.

T-Cell Receptor CDR3 Sequencing

Human CD3+ T cells were FACS-isolated from thymus of engrafted NSG mice.Purified DNA was subjected to next generation sequencing of thecomplementarity determining region 3 (CDR3) using immunoSEQ (AdaptiveBiotechnology, Seattle, Wash.) and analyzed with the immunoSEQ Analyzersoftware (Adaptive Biotechnology).

Affymetrix SNP 6.0 Genotyping of Engrafted Cells

250 ng aliquots of genomic DNA from human CD45+ BM cells (CD45 PE-Cy5(J33; Coulter)) from engrafted NSG mice and original PSCs (2 biologicalreplicates) were digested with either Nsp1 or Sty1. A universal adaptoroligonucleotide was then ligated to the digested DNAs. The ligated DNAswere diluted with water and three 10 uL aliquots from each well of theSty 1 plate and four 10 uL aliquots from each well of the Nsp 1 platewere transferred to fresh 96-well plates. PCR master mix was added toeach well and the reactions cycled as follows: 94° C. for 3 min; 30cycles of 94° C. for 30 s, 60° C. for 45 s, 68° C. for 15 s; 68° C. for7 min; 4° C. hold. Following PCR, the 7 reactions for each sample werecombined and purified by precipitation from 2-propanol/7.5M ammoniumacetate. The UV absorbance of the purified PCR products was measured toinsure a yield ≥4 ug/uL. 45 uL (≥180 ug) of each PCR product wasfragmented with DNAse 1 so the largest fragments were <185 bp. Thefragmented PCR products were then end-labeled with a biotinylatednucleotide using terminal deoxynucleotidyl transferase. Forhybridization, the end-labeled PCR products were combined withhybridization cocktail, denatured at 95° C. for 10 min and incubated at49° C. 200 mL of each mixture was loaded on a GeneChip and hybridizedovernight at 50° C. and 60 rpm. Following 16-18 hrs of hybridization,the chips were washed and stained using the GenomeWideSNP6_450 fluidicsprotocol with the appropriate buffers and stains. Following washing andstaining, the GeneChips were scanned on a GeneChip Scanner 3000 usingAGCC software. Genotype calls (chp files) were generated in AffymetrixGenotyping Console using the default parameters. The resulting chp fileswere analyzed for familial relationships using the identity by statealgorithm implemented in Partek Genomics Suite.

Pooled RNA-Sequencing

Engrafted human CD34+CD38−CD45+ HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then RNA was purified withRNeasy Micro kit (QIAGEN). QC of RNA was done by Bioanalyzer and Qubitanalysis. Passed samples were converted into library and sequenced byNextseq PE75 kit.

Single-Cell RNA-Sequencing with in-Droplet-Seq Technology

Engrafted human CD34+CD38−CD45+ HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then processed forin-droplet-barcoding according to a previous report⁶³. Library was QCedwith Bioanalyzer and sequenced by Nextseq PE 75 kit.

Lentiviral integration detection by ligation-mediated PCR andnext-generation sequencing

Either CD33+ myeloid, CD19+ B− and CD3+ T-cells were isolated from BMfrom HE-injected NSG mice. Genomic DNA was purified with QIAamp DNAMicro kit (QIAGEN). Ligation-mediated PCR-based detection of lentiviralintegration sites was done with Lenti-X Integration Site Analysis Kit(Clontech) according to manufacture's instruction. Sequencing-baseddetection (Integ-seq) was done following a previous report⁶⁴.

Single-Cell RNA-Sequencing with in-Droplet-Seq Technology

Engrafted human CD34+CD38−CD45+ HSCs were isolated from BM from eitheriPS-derived HE- or CB-injected NSG mice, then processed forin-droplet-barcoding according to a previous report 72. Library was QCedwith Bioanalyzer and sequenced by Nextseq PE 75 kit. The t-DistributedStochastic Neighbor Embedding (t-SNE) algorithm was used to visualizetranscriptome similarities and population heterogeneity of cord bloodHSCs and iPSC-derived HSCs. t-SNE performs a dimensionality reduction ofmultidimensional single-cell RNA-seq data into a low dimensional space,preserving pairwise distances between data-points as good as possible,allowing a global visualization of subpopulation structure and cell-cellsimilarities. We used the R package tsne in our analyses. The t-SNE mapwas initialized with point-to-point distances computed by classicalmultidimensional scaling and the R plot function was used to visualizet-SNE maps annotated by cord blood or iPSC-derived HSCs. Plots showingt-SNE maps colored by expression of selected genes were created usingthe ggplot2 package. For subpopulation identification, we used the top500 genes with highest variance to elucidate global differences amongsingle cells. To assess transcriptome similarities in terms of inductionof hematopoietic genes in iPSC-derived HSCs, we used 62 hematopoieticgenes for t-SNE analysis. GSEA

GSEA was performed with the desktop client version (javaGSEA, softwareis available at the Broad Institute website) with default parameters.RPKM values from the 7F-HSPC were obtained from the RNA seq (describedpreviously). These values were normalized to a terminally differentiatedcell (e.g., T-cells or B-cells) and the normalized values were used torank the most differentially expressed genes. These differentiallyexpressed genes were used to run GSEA with gene sets obtained frommSigDB (KEGG, Hallmark, immunological, transcription factors andchemical and genetic perturbations gene sets were used). In addition,gene sets specific to progenitors, cord blood or fetal-liver HSC wereobtained from previous reports⁷³ ²³. FDR <0.25 with a p-value of <0.05was considered significant.

Lentiviral Integration Detection by Ligation-Mediated PCR andNext-Generation Sequencing

Either CD33+ myeloid, CD19+ B− and CD3+ T-cells were isolated from BMfrom HE-injected NSG mice. Genomic DNA was purified with QIAamp DNAMicro kit (QIAGEN). Ligation-mediated PCR-based detection of lentiviralintegration sites was done with Lenti-X Integration Site Analysis Kit(Clontech) according to manufacture's instruction. Sequencing-baseddetection (Integ-seq) was done following a previous report⁷⁴.

Results

Cell identity is defined by gene regulatory networks that are governedby transcription factors (TFs)^(10,11). By supplying TFs that drivehematopoietic gene regulatory networks, several groups have generatedhematopoietic stem and progenitor cells from sources as diverse asfibroblasts, endothelial cells, and differentiated blood cells¹²⁻¹⁸. Ina prior study, we screened a set of 9 HSC-specific TFs for theirpotential to induce in vitro hematopoietic colony forming activity andin vivo engraftment from hPSC-derived myeloid cells, and isolated a setof five TFs (HoxA9, ERG, RORA, SOX4, and MYB) that promoted short-termengraftment of erythroid and myeloid cells, but did not achievelong-term multilineage hematopoiesis¹⁷. Recent approaches haverecapitulated HE differentiation from hPSCs to generate cells withmyeloid and T cell hematopoietic potential in vitro¹⁹⁻²¹; however, few,if any, cells capable of engrafting irradiated murine hosts weregenerated.

We adapted a protocol to derive HE from hPSCs and verified itshematopoietic potential²⁰. We isolated HE (characterized by thesemarkers: FLK1+CD34+CD43-CD235A−) at day 8 of embryoid body (EB)formation (data not shown), and upon further culture supplemented withhematopoietic cytokines observed the endothelial to hematopoietictransition. Consistent with previous reports^(19,20), we documented adecrease in expression of endothelial genes (YAP, FOXC1, COUPTFII), anincrease in levels of hematopoietic lineage genes (RUNX1, MYB, GATA2,SCL), and concomitant emergence of CD34+CD45+ hematopoietic cells (datanot shown). However, multiple attempts to engraft irradiated immunedeficient recipients with these cultured cells failed.

We hypothesized that introduction of HSC-specific TFs would endowhPSC-derived HE with the potential to engraft multi-lineagehematopoiesis in vivo. We queried the HE by CellNet, a cell-typeclassification algorithm that compares in vitro derived cells against apanel of comparator cell types²². HE was classified as predominantlyendothelium with partial identity to hematopoietic stem and progenitorcells (HSPCs; data not shown). To identify TFs likely to specify HSPCfate, we reasoned that functionally relevant TFs would be evolutionarilyconserved. Thus, we used two independent mouse^(23,24) and twohuman^(25,26) datasets to select 12 TFs enriched in fetal liver-HSCs(FL-HSCs) relative to HE (data not shown), and selected other candidatesfrom prior reports that used TFs to covert endothelial cells″,hPSC-derived myeloid cells¹⁷, or committed lymphoid cells¹² tohematopoietic progenitor cells. For the data set, comparison of theexpression profile of HSC-specific TFs between HE(CD34+FLK1+CD43−CD235A−) vs FL-HSCs (CD34+CD38−CD90+CD45+) were made. 12HSC-specific TFs were enriched in FL-HSCs and downregulated in HE. ThoseTFs were cloned to Dox-inducible lentiviral vector. The expression levelof SOX17, a marker of HE, was 2.4-fold higher in HE (N=7) than FL-HSCs(N=10). * P<0.001. All together, we assembled a library of 26 TFs, whichwe cloned into a doxycycline-inducible lentiviral vector (FIG. 1A). Weinfected the library into HE at day 3 of endothelial-to-hematopoietictransition (EHT) culture (efficiency was >50%; FIGS. 1A and 4). Weinjected the transduced cells intrafemorally into sublethally irradiatedimmune-deficient NOD/SCID/Gamma (NSG) mice. Mice received doxycycline intheir drinking water and diet for two weeks to induce transgeneexpression¹², after which doxycycline was withdrawn. We observed humanCD45+ cells in peripheral blood of injected mice up to 14 weeks,indicating long-term hematopoietic engraftment (FIG. 1B). Examination ofBM and thymus demonstrated the presence of human erythroid (GLY-A+),myeloid (CD33+), B (CD19+) and T-cells (CD3+) (FIG. 1C). When human cordblood (CB)-HSCs engraft in NSG mice, a predominance of B-cells over Tcells is typical²⁷. Analysis of BM from 3 of 6 recipients engrafted withhiPSC-derived HE and 2 of 5 recipients engrafted with hESC-derived HE at10-14 weeks showed notable reconstitution of T-cells and/or B-cells,comprising 46±20% and 37±9.7% proportion of human grafts, compared withCB-HSC derived grafts, in which B-cells comprised 86±11% of engraftedhuman cells (FIG. 1C). Intrafemoral injection of HSCs into one femurrepopulates the contralateral femur through expansion and homing ofHSCs²⁸. Notably, following unilateral intrafemoral injection oftransduced HE, we observed engraftment in both femurs. Engraftment ofhuman CD45+ cells in BM averaged 2.5±3.4% from transduced HE, comparedwith 46±18% from CB-HSCs. To confirm the hPSC origin of our engraftedcells, we demonstrated by SNP array genotyping that human CD45+ cellscollected from peripheral blood were identical to the input hPSCs (datanot shown). Together, these results demonstrate that infection with a26-TF library enables multi-lineage hematopoietic engraftment fromhPSC-derived HE.

We then determined which of the 26 TFs could be detected in theengrafted human cells by PCR amplification in sorted populations ofCD33+ myeloid, CD19+ B cell, and CD3+ T cells. 7 TFs (ERG, HOXA5, HOXA9,HOXA10, LCOR, RUNX1, SPI1) were consistently detected in myeloid, B- andT-cells of five engrafted recipients, indicating that these factorsconferred multipotency. FIG. 5 shows the Venn diagram of expressionprofile of TFs during hematopoietic ontogeny. ZKSCAN1, SSBP2, MAFF,DACH1, and SOX4 were detected in certain infected cultures but notconsistently across all animals, perhaps reflecting their potential toenhance engraftment under some experimental conditions, or perhapsindicating that these genes are passengers.

We next determined whether the 7 common TFs were sufficient to supportmultilineage engraftment of HE in vivo. We transduced HE with these 7TFs, injected cells intrafemorally into sublethally irradiated NSG mice,and treated with doxycycline for 2 weeks. Chimerism of human CD45+ inmurine BM at 8 weeks was 1.9±1.8% for library transduced cells, 12±5.1%for cells transduced with the defined 7 TFs, and 43±4.2% for CB-HSCs(FIGS. 1B and 1D), reflecting considerably enhanced engraftmentpotential for the 7 TFs. We sought to determine the minimal combinationof TFs required for multilineage engraftment by a factor-minus-one (FMO)approach. Exclusion of individual factors did not ablate engraftment,though RUNX1, ERG, LCOR, HOXA5 or HOXA9 omission reduced chimerism in BMmost significantly at 8 weeks (FIG. 2B). These data indicate that at aminimum, RUNX1, ERG, LCOR, HOXA5 and HOXA9 facilitate optimalengraftment.

We monitored mice engrafted with HE transduced with the defined 7 TFsuntil 12 weeks. 2 out of 5 recipients had multi-lineage engraftment witherythroid (GLY-A+), myeloid (CD33+), B-cells (CD19+) and T-cells (CD3+)(FIGS. 1D and 1F). The 3 other recipients had both B-cells and T-cellsand either erythroid or myeloid cells (FIG. 1F). We next validated theself-renewal capacity of HE-derived cells by secondary transplantation.2 out of 5 recipients engrafted with multilineage erythroid,neutrophils, B-cells and T-cells at 8 weeks (FIG. 2C). The percentage ofphenotypic HSCs (CD34+CD38−) was lower in secondary than primary mice,indicating depletion of the HSC-like population over time, however,multilineage engrafts were still observed over 14 weeks (FIG. 2D).

To determine which of 7 TFs were consistently isolated in multiplelineages of secondary engrafted mice, we performed PCR amplification ofmyeloid, B and T cells from 2 mice and detected LCOR, HOXA5, HOXA9 andRUNX1 in every lineage, while ERG was noted in only myeloid and B-cells.SPI1 and HOXA10 appeared dispensable (FIG. 2A). We then investigated ifERG, LCOR, HOXA5, HOXA9 and RUNX1 are essential to confer functionalhematopoiesis on iPS-HE. 5 factors (LCOR-HOXA5-HOXA9/RUNX1-ERG) wereinduced by polycistronic lentiviral vectors and conferred multilineageerythroid, neutrophils, B-cells (including plasmacytoid lymphocytes) andT-cells at 12 weeks (FIGS. 1E and 1F).

The RUNX1 TF is well known to facilitate EHT²⁹. We determined if definedTFs enhance EHT using the RUNX1+24 enhancer-tdTomato reporter thatactivates during hematopoietic cell emergence from HE³⁰. Upon expressionof 7 TFs in HE, the reporter was induced 2.4-fold compared to control,correlating with an increase in hematopoietic genes (MYB, HDAC1, GATA2)(data not shown). These data suggest that the 7 TFs we have identifieddrive hematopoiesis in part through facilitating Runx1-mediated EHT.

A previous report demonstrated the induction of engraftable progenitorcells from hESCs, however, it is uncertain if myeloid and lymphoidprogeny came from monoclonal origin that is feature of stem cells, ordistinctly committed progenitor cells¹⁸. We then determined whether themyeloid and lymphoid progeny of the iPS-derived HE transduced with the 7TFs were of monoclonal origin by comparing lentiviral integrationpatterns. We isolated genomic DNA from myeloid (CD33+), B-(CD19+) andT-cells (CD3+) from BM and thymus at 10 weeks post transplantation,followed by adaptor-ligation PCR to detect lentiviral integrationsequences. Common integration sequences were detected in myeloid,B-cells, and T-cells in each individual recipient (data not shown),consistent with a monoclonal origin of at least some of the cells.Remarkably, T-cells had several unique minor integrations indicating thepresence of a diverse human T-cell pool in engrafted NSG, as reportedpreviously³¹. These data demonstrate that in vivo screening identified acore set of between 5-7 TFs that induce HSC-like cells capable ofrepopulating irradiated primary and secondary mice with multi-lineagehematopoiesis.

Although some recipient mice manifest clonal multi-lineage hematopoiesisindicative of reconstitution from HSC-like cells, secondary mice showeda reduced frequency of CD34+38− phenotypic HSCs. The frequency ofphenotypic HSCs in BM was 2.0% (CB-HSC engrafted group) versus 0.47% (HEengrafted group) (FIG. 2F). The cycling state of 7 TF HSPCs (based onKi67 expression) was significantly higher than that of CB-HSCs insecondary recipients (FIG. 2G). Human CD45+ cells recovered fromengrafted BM revealed residual transgene expression despite cessation ofdoxyxcyline (data not shown). Interestingly, LCoR has been reported tobe a negative regulator of p21 expression³², and p21-deficient HSCs areknown to undergo exhaustion due to persistent cycling³³. Thus, wespeculate that trace levels of residual transgene expression couldcompromise the long term cycling of the HSPC-like cells.

We examined erythroid, myeloid, and lymphoid cells recovered fromengrafted mice, and compared their functional properties toCB-HSC-derived cells. Definitive erythropoiesis is characterized byglobin switching and enucleation³⁴ ³⁵. Most erythroid cells generatedfrom hPSCs express embryonic and fetal globins and retain nuclei¹⁷. Thehuman erythroid cells recovered from engrafted mice lacked expression ofembryonic HBE, and expressed fetal HBG and adult HBB at levelscomparable to CFU-E from human CB (FIG. 3A). Remarkably, ¼ of humanGLY-A+ cells derived from the 7TF HSPCs were enucleated (FIG. 3B). Humanmyeloid cells in NSG recipients respond to cytokine stimuli byactivation of myeloperoxidase (MPO)³⁶. We isolated CD45+CD15+ PECAM+neutrophils from engrafted BM at 8 weeks, and compared MPO production toengrafted CB-derived cells. PMA stimulation enhanced the production ofMPO 3.0-fold relative to unstimulated neutrophils (FIG. 3C). Bona fidehuman HSCs generate functional T- and B-cells in NSG mice²⁷. IgM and IgGantibody could be detected in the serum of NSG mice engrafted with 7 TFHSPCs, indicating the cooperative activity of T and B cells in mediatingimmunoglobulin class switching, secretion and boosted by Ovalbumin (OVA)(FIGS. 3D and 3E). We isolated mature CD3+ T-cells from BM and measuredinterferon γ (IFNγ) production. Notably, this CD3+ population expressedCD4, and not CD8. PMA/Ionomycin (PMAI)-stimulation enhanced IFNγproduction 3.0-fold in 7 TF CD3+ cells vs 4.4-fold in CB-derivedCD3+(FIG. 3F). T-cells develop from CD4−CD8− double-negative cellsfollowed by CD4+CD8+ double-positive cells that express surface TCR/CD3complex, which differentiate to either CD4 or CD8 single-positive Tcells in thymus, which migrate to blood and BM37. At 8 weeks, 7 TFHSPC-derived thymocytes were predominantly CD4+CD8+(55±22%), with fewCD4+CD8− (1.8±0.42%) and CD4-CD8+(0.80±0.36%). Human CD3+ T-cellsdifferentiated from HSCs in NSG possess either TCRαβ (>60%) or TCRγδ(<30%)²⁷, consistent with our observation that 7 TF HSPC-derived graftsproduced both TCRαβ (89±9.3%) and TCRγδ (3.8±6.6%) cells (FIG. 3G).Development of a diverse population of antigen-specific T cells requiresrearrangement of germline-encoded TCR genes″, largely mediated by thecomplementarity determining region 3 (CDR3) within variable (V) genesegments of the TCRA and TCRB genes³⁹. To determine clonotype diversity,we profiled the CDR3 region of TCRB on CD3+ T-cells in reconstitutedmice using next generation sequencing. We observed a high degree ofcombinatorial diversity in the V gene segment in CD3+ T-cells isolatedfrom either CB-engrafted NSG or 7 TF HSPC-engrafted NSG mice with theCDR3 length following a standard Gaussian distribution (FIG. 3H).

The generation of functional HSC-like cells from PSCs has been a longsought goal in hematology research. By directed differentiation of hPSCsto HE followed by in vivo screening of TFs for hematopoietic progenitorspecification, we identified 7 TFs that together confer HSC-likeengraftment, self-renewal and multilineage capacity. Considerable workremains to establish engraftment with transgene-free cells, and toachieve stable, long-term multi-lineage hematopoietic chimerism inhumanized mice. Such a system will facilitate modeling a multitude ofgenetic blood disorders that are not faithfully recapitulated ingenetically engineered murine models, and for which adequate marrowsamples are not readily obtained, as for patients with bone marrowfailure syndromes.

Combinations of TFs have been introduced into differentiated bloodcells¹² ¹⁷ to endow HSC-like properties in a murine system¹², buttransplantable human HSCs with multilineage capacity have to date notbeen derived from hPSCs. Recent advances in the directed differentiationof PSCs to definitive HE have provided a supportive context forscreening of HSC-specifying TFs. Each of the identified TFs in our studyplays a role in HSC development, maintenance of long-term HSCs, orlineage commitment. RUNX1 is essential for hematopoietic commitment ofHE and can convert endothelial cells to hematopoietic progenitor cells¹³⁴⁰ ⁴¹. LCOR, a component of histone deacetylation complex, is mutated inB-cell lymphoma, suggesting a role in B-lymphopoiesis⁴² ⁴³, but thisfactor has not previously been implicated in HSC functions and its roleremains to be defined. SPI1 (also known as PU.1) is required forhematopoietic progenitor cell emergence and regulates myeloidspecification⁴⁴ ⁴⁵ ⁴⁶. HOX family members have been reproduciblyimplicated in hematopoiesis across species^(17,47,48). HOXA9 is the keyhomeotic gene that defines HSC identity^(49,50), interacting with ERG tosupport HSC renewal during embryogenesis and stress hematopoiesis⁵¹⁻⁵³,suggesting a basis for the functional cooperation of HOXA9 and ERG inour system. HOXA10 augments induction of erythroid cells from ESCs⁵⁴,but appears to be at least partially dispensable in our system. Ectopicexpression of HOXA5 induces commitment of HSCs to myeloid lineages⁵⁵.HOXA5 is a transcriptional target of Notch signaling in T-cellprogenitors along with HOXA9 and HOXA10, consistent with a role inT-lymphopoiesis⁵⁶. These factors share binding sites in the genome andcooperate to recruit chromatin modulators (e.g. RUNX1 and HOXAfamilies)^(53,57) to induce and maintain HSPCs.

Our FMO approach to the defined 7 TFs indicated that they are part of acommon gene regulatory network with some redundancy, as exclusion ofindividual factors did not fully abrogate engraftment of HE. Thepossibility remains that 7 TF HSPCs are predominantly fetal as shown inhESC-derived hematopoietic cells in a previous study⁵⁸, supported bytheir rapid cycling state, and predominance of CD4+CD8+ T cells in thethymus.

Our study suggests that we are ever closer to realizing the potential ofderivation of HSC-like cells from renewable sources like pluripotentstem cells. Such cells, when derived from patients with genetic blooddisorders, offer considerable promise for modeling human blood disease,for humanizing mice for research applications, and for testing thecapacity of gene therapy vectors or pharmacologic agents to restorehematopoietic function. The long term goal remains the derivation ofbona fide transgene-free HSCs for applications in research and therapy.

The references cited herein and throughout the specification areincorporated herein by reference.

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What is claimed is:
 1. A method for making human hematopoietic stemcells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) forcellular replacement therapy, the method comprising in vitrotransfection of human hemogenic endothelial cells (HE) with an exogenousgene coding copy of each of the following transcription factorsETS-related gene (ERG), Homeobox protein A9 (HOXA9), Homeobox protein A5(HOXA5), Ligand Dependent Nuclear Receptor Corepressor (LCOR) and RuntRelated Transcription Factor 1 (RUNX1), wherein the human HE cells arederived from embryoid bodies (EB) produced from human iPSCs or humanESCs, wherein the transcription factors are expressed in the transfectedhuman HE cells, thereby producing a population of multilineage humanHSCs and HSPCs for cellular replacement therapy.
 2. The method of claim1, further comprising, prior to in vitro transfection, the steps of: a.generating embryonic bodies (EB) from human pluripotent stem cells; andb. isolating human hemogenic endothelial cells (HE) from the resultantpopulation of EB.
 3. The method of claim 2, wherein the EB are generatedor induced from human pluripotent stem cells (PSC) by culturing orexposing the PSC to morphogens for about 8 days.
 4. The method of claim3, wherein the population of human PSC is a population of human inducedpluripotent stem cells (iPS cells) or human embryonic stem cells (ESC).5. The method of claim 4, wherein the induced pluripotent stem cells areproduced by introducing reprogramming factors OCT4, SOX2, KLF4 andoptionally (i) c-MYC or (ii) nanog and LIN28 into human somatic cells.6. The method of claim 5, wherein the human somatic cells are selectedfrom the group consisting of peripheral blood circulating cells, Blymphocytes (B-cells), T lymphocytes, (T-cells), fibroblasts, andkeratinocytes.
 7. The method of claim 2, wherein the EBs are: a. lessthan 800 microns in size and are selected; b. are compactly adhered toeach other and requires trypsin digestion in order to dissociate thecells to individual cells; and/or c. are dissociated prior to theisolation of HE.
 8. The method of claim 1, wherein the HE are: a.isolated immediately from selected and dissociated EB; and/or b. FLK1+,CD34+, CD43−, and CD235A−.
 9. The method of claim 1, wherein themultilineage human HSCs are CD34+CD38−CD45+.
 10. The method of claim 1,wherein the multilineage human HSPCs and/or hematopoietic cells areCD34+CD45+.
 11. An engineered cell or population of engineered cellsderived from a population of human HE and produced by the method ofclaim
 1. 12. A composition comprising the population of engineered cellsof claim
 11. 13. A pharmaceutical composition comprising the populationof engineered cells of claim 11 and a pharmaceutically acceptablecarrier.
 14. The method of claim 1, further comprising the step ofinducing endothelial-to-hematopoietic transition (EHT) in culture in thetransfected HE to obtain human hematopoietic stem cells, wherein theendothelial-to-hematopoietic transition occurs by culturing the isolatedHE in the presence of thrombopoietin (TPO), interleukin (IL)-3, stemcell factor (SCF), IL-6, IL-11, insulin-like growth factor (IGF)-1,erythropoietin (EPO), vascular endothelial growth factor (VEGF), basicfibroblast growth factor (bFGF), bone morphogenetic protein (BMP)4, Fmsrelated tyrosine kinase 3 ligand (Flt-3L), sonic hedgehog (SHH),angiotensin II, and chemical AGTR1 (angiotensin II receptor type I)blocker losartan potassium.